Trifunctional constructs with tunable pharmacokinetics useful in imaging and anti-tumor therapies

ABSTRACT

The present technology provides compounds, as well as compositions including such compounds, useful for imaging and/or treatment of a glioma, a breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lung cancer, a small cell lung cancer, a bladder cancer, a colon cancer, a primary, gastric adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell carcinoma, and/or a prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.16/134,789, filed Sep. 18, 2018, which is a Continuation-in-Part of U.S.patent application Ser. No. 15/630,808, filed Jun. 22, 2017, now U.S.Pat. No. 10,179,117, which claims the benefit of U.S. Provisional PatentApplication No. 62/353,735, filed on Jun. 23, 2016. U.S. patentapplication Ser. No. 16/134,789 is also a Continuation-in-Part ofInternational Patent Application No. PCT/US2018/026340, filed Apr. 5,2018, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/482,038, filed Apr. 5, 2017, and U.S. Provisional PatentApplication No. 62/574,720, filed Oct. 19, 2017, the entire disclosuresof each of which are incorporated herein by reference for any and allpurposes in their entirety.

U.S. GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant numberUL1TR00457 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

The present technology relates to targeted drug delivery, moreparticularly compounds useful in anti-tumor therapy. For example, thecompositions described herein may be used as radiotherapy agents for thetreatment of cancers such as prostate cancer.

SUMMARY

The present invention details compounds that are useful agents for theradiotherapy of tumors. The compounds have affinity for cellular markersassociated with neoplastic tissues as well as blood proteins, therebyproviding increased residence time in the circulatory system of arecipient, which increases tumor perfusion and achieves greater uptakeand compound loading of the tumor, as well as minimizing compoundbinding to non-target tissues with consequent side effects. In oneaspect of the present technology, a multi-targeted compound is providedthat includes a tumor-binding domain, an albumin-binding domain, and acytocidal or cytostatic therapeutic agent, where the tumor-bindingdomain includes an active site that is distal to and stericallyunimpeded by the albumin-binding domain and the therapeutic agent, andthe relative affinity of the tumor-binding domain and thealbumin-binding domain differ in specific affinity by a factor of atleast 100 to about 10,000.

In a related aspect, a multi-targeted compound is provided, having aplurality of sterically unimpeded targeting domains, where the compoundincludes a first targeting domain that includes a blood-protein bindingdomain having specific affinity for binding human serum albumin in therange of about 0.25 to 50 micromolar, a second targeting domain thatincludes a tumor-binding domain having specific affinity for a tumorassociated molecular target in the range of about 0.1 to 75 nanomolar;wherein the relative affinities of the first and second targetingdomains differ in specific affinity by a factor of at least 100 to about10,000; and a therapeutic domain that includes a cytocidal or cytostatictherapeutic agent. In a more specific embodiment of the above, theinvention provides for a compound including a multi-targeted agenthaving a plurality of sterically unimpeded targeting domains comprising,a first targeting domain comprising a blood-protein binding domainhaving specific affinity for binding human serum albumin in the range ofabout 0.25 to 50 micromolar, a second targeting domain comprising atumor-binding domain having specific affinity for PSMA in the range ofabout 0.1 to 75 nanomolar; wherein the relative affinities of the firstand second targeting domains differ in specific affinity by a factor ofat least 100 to about 10,000; and a therapeutic domain comprising acytocidal or cytostatic therapeutic agent. In various embodiments, thetumor associated molecular target is selected from one or more of atumor-specific cell surface protein, prostate specific membrane antigen(PSMA), somatostatin peptide receptor-2 (SSTR2), alphavbeta3 (αvβ3),alphavbeta6, a gastrin-releasing peptide receptor, a seprase, fibroblastactivation protein alpha (FAP-alpha), an incretin receptor, aglucose-dependent insulinotropic polypeptide receptor, VIP-1, NPY, afolate receptor, LHRH, a neuronal transporter (e.g., noradrenalinetransporter (NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4,ED2,TF-antigen, an endothelial specific marker, neuropeptide Y, uPAR,TAG-72, a claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specificcell surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, oran overexpressed peptide receptor. The compounds include a tumor-bindingdomain that binds to the tumor associated molecular target with moderateto high affinity (e.g. an affinity constant (K_(D)) of approximately10{circumflex over ( )}-8M to 10{circumflex over ( )}-10M). In variousembodiments, the compounds include a cytocidal or cytostatic therapeuticagent that is a toxin, a venom, a metabolic poison, a chemotherapeuticagent, an auger electron-emitting radionuclide, a beta-emittingradionuclide, or an alpha-emitting radionuclide. In other suchembodiments, the therapeutic domain comprises a covalently conjugatedchelating agent or a covalently conjugated polyaza polycarboxylicmacrocycle. In still other embodiments, the therapeutic domain furthercomprises an auger electron-emitting radionuclide, a beta-emittingradionuclide, or an alpha-emitting radionuclide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides PET images of mice injected with ⁶⁸Ga-RPS-055 (top) or⁶⁸Ga-DKFZ-617 (bottom) both 1 h and 3 h post injection. All images areof the same mouse bearing a small LNCaP tumor in the right shoulder(arrow). Images were taken on sequential days using a Siemens InveonμPET/μCT system (Siemens Corp., Munich, Germany). All images werewindowed to a maximum of 4% injected dose per cc tissue and are decaycorrected and dose corrected.

FIG. 2 provides PET images of mice injected with ⁶⁸Ga-RPS-055 (leftpanels) or ⁶⁸G-RPS-056 (right panels) both 1 h (top) and 3 h (bottom)post injection. All images are of the same mice bearing small LNCaPtumors in their right shoulders. Images were taken on sequential daysusings a Siemens Inveon μPET/μCT system (Siemens Corp., Munich,Germany). All images were windowed to a maximum of 20% injected dose percc tissue and are decay corrected and dose corrected.

FIG. 3 provides SPECT images of mice injected with ¹⁷⁷Lu-RPS-055 both 4h (top) and 3 h (bottom) post injection. All images are of the same micebearing small LNCaP tumors in their right shoulders (left panels andright panels). Images were taken on the same day using a Siemens InveonμSPECT/μCT system (Siemens Corp., Munich, Germany).

FIG. 4 shows PET images of BALB/C nu/nu mice bearing LNCaP tumorxenografts and injected intravenously with either ⁶⁸Ga-RPS-061,⁶⁸Ga-PSMA-617 or ⁶⁸Ga-RPS-030. The mice were imaged on sequential daysat 1 h (left) and 3 h (right) post injection using a Siemens InveonμSPECT/μCT system (Siemens Corp., Munich, Germany).

FIGS. 5A-C provide histograms illustrating the biodistribution of²²⁵Ac(NO₃)₃ (FIG. 5A), [²²⁵Ac(macropa)]⁺ (FIG. 5B), and [²²⁵Ac(DOTA)]⁻(FIG. 5C) for select organs following intravenous injection in mice.Adult C57BL/6 mice were sacrificed 15 min, 1 h, or 5 h post injection.Values for each time point are given as mean % ID/g 1±SD.

FIG. 6 illustrates the biodistribution of ²²⁵Ac-macropa-RPS-070following intravenous injection in LNCaP tumor xenograft mice. Mice weresacrificed 4, 24, or 96 h post injection. Values for each time point aregiven as mean % ID/g 1 SEM.

FIG. 7 provides PET images of LNCaP xenograft mice with ⁶⁶Ga-labeledtracers at 1 h, 3 h, 6 h and 24 h post injection. Mice were injectedintravenously with a bolus injection of 0.56-5.4 MBq (15-145 μCi) of thetracer. The total mass of ligand injected was 4 μg. Prior to imaging,the mice were anesthetized with isoflurane and then imaged for 30 min.The images were corrected for decay and for activity injected.

FIG. 8 provides the biodistribution of ¹⁷⁷Lu-RPS-068, ¹⁷⁷Lu-RPS-063,¹⁷⁷Lu-RPS-061, ¹⁷⁷Lu-RPS-069, ¹⁷Lu-RPS-066, ¹⁷Lu-RPS-067 and¹⁷Lu-PSMA-617. Male athymic nude mice bearing LNCaP xenograft tumors(n=5 per time point) were injected intravenously with 348-851 kBq(9.4-23 μCi) of the labeled compound and sacrificed at 4 h, 24 h and 96h p.i. The total mass of ligand injected was 37-50 ng (23-25 μmol).

FIG. 9 provides a comparison of the blood pool activity of different¹⁷⁷Lu-labeled ligands at 4 h, 24 h and 96 h post injection. Errors areexpressed as SEM. RPS ligands are displayed in order of increasing size.

FIG. 10 provides time-activity curves (TAC) of tumor and kidney uptakeof ¹⁷⁷Lu-PSMA-617, ¹⁷⁷Lu-RPS-061, ¹⁷⁷Lu-RPS-063, ¹⁷⁷Lu-RPS-066,¹⁷⁷Lu-RPS-067, ¹⁷⁷Lu-RPS-068 and ¹⁷⁷Lu-RPS-069 in male athymic nude micebearing LNCaP xenograft tumors. Uptake is expressed as % ID/g.

FIG. 11 provides a comparison of relative dose integral in the tumor ofmale LNCaP xenograft tumor-bearing mice injected with the corresponding¹⁷⁷Lu-labeled compounds and studied over 96 h. Values are normalized to¹⁷⁷Lu-PSMA-617.

FIG. 12 shows the uptake of activity in blood, normal tissue and tumorin male BALB/C nu/nu mice bearing LNCaP xenograft tumors. Mice (n=4/timepoint) were injected intravenously with 105 kBq ²²⁵Ac-RPS-074 andsacrificed at 4 h, 24 h, 7 d, 14 d and 21 d p.i.

FIG. 13 plots the change in average tumor volumes of individual maleBALB/C nu/nu mice bearing LNCaP xenograft tumors and treated with a) 138kBq ²²⁵Ac-RPS-074; b) 74 kBq ²²⁵Ac-RPS-074; c) 37 kBq ²²⁵Ac-RPS-074; d)133 kBq ²²⁵Ac-DOTA-Lys-IPBA; and e) vehicle.

FIG. 14 provides ⁶⁸Ga-PSMA-11 μPET/CT images of mice treated with 138kBq 2²⁵Ac-RPS-074 (left) or 74 kBq ²²⁵Ac-RPS-074 (right). Images wereacquired 60 min post injection and are corrected for decay and foractivity injected.

FIG. 15 plots a Kaplan-Meier curve illustrating the overall survival ofthe mice. ##=Activity of ²²⁵Ac-DOTA-Lys-IPBA administered. Mice weresacrificed when tumor volume exceeded 2000 mm³.

DETAILED DESCRIPTION

The following terms are used throughout as defined below.

As used herein and in the appended claims, singular articles such as “a”and “an” and “the” and similar referents in the context of describingthe elements (especially in the context of the following claims) are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the claims unless otherwise stated. No language in the specificationshould be construed as indicating any non-claimed element as essential.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³²and S³⁵ are thus within the scope of the present technology. Proceduresfor inserting such labels into the compounds of the present technologywill be readily apparent to those skilled in the art based on thedisclosure herein.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup is substituted with one or more substituents, unless otherwisespecified. In some embodiments, a substituted group is substituted with1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groupsinclude: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy,aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy,and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters;urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols;sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e.,SF₅), sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and ring systemsin which a bond to a hydrogen atom is replaced with a bond to a carbonatom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

As used herein, C_(m)-C_(n), such as C₁-C₁₂, C₁-C₈, or C₁-C₆ when usedbefore a group refers to that group containing m to n carbon atoms.

Alkyl groups include straight chain and branched chain alkyl groupshaving from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms.Examples of straight chain alkyl groups include groups such as methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octylgroups. Examples of branched alkyl groups include, but are not limitedto, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl,and 2,2-dimethylpropyl groups. Alkyl groups may be substituted orunsubstituted. Representative substituted alkyl groups may besubstituted one or more times with substituents such as those listedabove, and include without limitation haloalkyl (e.g., trifluoromethyl),hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups havingfrom 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocycliccycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In someembodiments, the cycloalkyl group has 3 to 8 ring members, whereas inother embodiments the number of ring carbon atoms range from 3 to 5, 3to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridgedcycloalkyl groups and fused rings, such as, but not limited to,bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Cycloalkylgroups may be substituted or unsubstituted. Substituted cycloalkylgroups may be substituted one or more times with, non-hydrogen andnon-carbon groups as defined above. However, substituted cycloalkylgroups also include rings that are substituted with straight or branchedchain alkyl groups as defined above. Representative substitutedcycloalkyl groups may be mono-substituted or substituted more than once,such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstitutedcyclohexyl groups, which may be substituted with substituents such asthose listed above.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, andtypically 4 to 10 carbon atoms. Cycloalkylalkyl groups may besubstituted or unsubstituted. Substituted cycloalkylalkyl groups may besubstituted at the alkyl, the cycloalkyl or both the alkyl andcycloalkyl portions of the group. Representative substitutedcycloalkylalkyl groups may be mono-substituted or substituted more thanonce, such as, but not limited to, mono-, di- or tri-substituted withsubstituents such as those listed above.

Alkenyl groups include straight and branched chain alkyl groups asdefined above, except that at least one double bond exists between twocarbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group hasone, two, or three carbon-carbon double bonds. Examples include, but arenot limited to vinyl, allyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂,—C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, among others. Alkenyl groups may besubstituted or unsubstituted. Representative substituted alkenyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di- or tri-substituted with substituents such asthose listed above.

Cycloalkenyl groups include cycloalkyl groups as defined above, havingat least one double bond between two carbon atoms. Cycloalkenyl groupsmay be substituted or unsubstituted. In some embodiments thecycloalkenyl group may have one, two or three double bonds but does notinclude aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbonatoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbonatoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenylgroups include cyclohexenyl, cyclopentenyl, cyclohexadienyl,cyclobutadienyl, and cyclopentadienyl.

Cycloalkenylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of the alkyl group is replaced with a bond to acycloalkenyl group as defined above. Cycloalkenylalkyl groups may besubstituted or unsubstituted. Substituted cycloalkenylalkyl groups maybe substituted at the alkyl, the cycloalkenyl or both the alkyl andcycloalkenyl portions of the group. Representative substitutedcycloalkenylalkyl groups may be substituted one or more times withsubstituents such as those listed above.

Alkynyl groups include straight and branched chain alkyl groups asdefined above, except that at least one triple bond exists between twocarbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group hasone, two, or three carbon-carbon triple bonds. Examples include, but arenot limited to —C≡CH, —C≡CCH₃, —CH₂C≡CCH₃, —C≡CCH₂CH(CH₂CH₃)₂, amongothers. Alkynyl groups may be substituted or unsubstituted.Representative substituted alkynyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted with substituents such as those listed above.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups herein include monocyclic, bicyclic andtricyclic ring systems. Thus, aryl groups include, but are not limitedto, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl,anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In someembodiments, aryl groups contain 6-14 carbons, and in others from 6 to12 or even 6-10 carbon atoms in the ring portions of the groups. In someembodiments, the aryl groups are phenyl or naphthyl. Aryl groups may besubstituted or unsubstituted. The phrase “aryl groups” includes groupscontaining fused rings, such as fused aromatic-aliphatic ring systems(e.g., indanyl, tetrahydronaphthyl, and the like). Representativesubstituted aryl groups may be mono-substituted or substituted more thanonce. For example, monosubstituted aryl groups include, but are notlimited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups,which may be substituted with substituents such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 16carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Aralkylgroups may be substituted or unsubstituted. Substituted aralkyl groupsmay be substituted at the alkyl, the aryl or both the alkyl and arylportions of the group. Representative aralkyl groups include but are notlimited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkylgroups such as 4-indanylethyl. Representative substituted aralkyl groupsmay be substituted one or more times with substituents such as thoselisted above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi-and tricyclic rings having 3 to 16 ring members, whereas other suchgroups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members.Heterocyclyl groups encompass aromatic, partially unsaturated andsaturated ring systems, such as, for example, imidazolyl, imidazolinyland imidazolidinyl groups. The phrase “heterocyclyl group” includesfused ring species including those comprising fused aromatic andnon-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. Heterocyclyl groups may besubstituted or unsubstituted. Heterocyclyl groups include, but are notlimited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl,dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl,imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl,oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl,pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl,dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl,quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl(pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl,benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl,benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl,benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl),triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl,guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl,thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl,dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl,tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl,tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl,tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, andtetrahydroquinolinyl groups. Representative substituted heterocyclylgroups may be mono-substituted or substituted more than once, such as,but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-,5-, or 6-substituted, or disubstituted with various substituents such asthose listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl(pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl(azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl,benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl,adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fusedring compounds in which all rings are aromatic such as indolyl groupsand include fused ring compounds in which only one of the rings isaromatic, such as 2,3-dihydro indolyl groups. Heteroaryl groups may besubstituted or unsubstituted. Thus, the phrase “heteroaryl groups”includes fused ring compounds as well as includes heteroaryl groups thathave other groups bonded to one of the ring members, such as alkylgroups. Representative substituted heteroaryl groups may be substitutedone or more times with various substituents such as those listed above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheterocyclyl group as defined above. Heterocyclylalkyl groups may besubstituted or unsubstituted. Substituted heterocyclylalkyl groups maybe substituted at the alkyl, the heterocyclyl or both the alkyl andheterocyclyl portions of the group. Representative heterocyclyl alkylgroups include, but are not limited to, morpholin-4-yl-ethyl,furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl,tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representativesubstituted heterocyclylalkyl groups may be substituted one or moretimes with substituents such as those listed above.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above. Heteroaralkyl groups may besubstituted or unsubstituted. Substituted heteroaralkyl groups may besubstituted at the alkyl, the heteroaryl or both the alkyl andheteroaryl portions of the group. Representative substitutedheteroaralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the presenttechnology are designated by use of the suffix, “ene.” For example,divalent alkyl groups are alkylene groups, divalent aryl groups arearylene groups, divalent heteroaryl groups are divalent heteroarylenegroups, and so forth. Substituted groups having a single point ofattachment to the compound of the present technology are not referred tousing the “ene” designation. Thus, e.g., chloroethyl is not referred toherein as chloroethylene. Such groups may further be substituted orunsubstituted.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Alkoxy groups may besubstituted or unsubstituted. Representative substituted alkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The terms “alkanoyl” and “alkanoyloxy” as used herein can refer,respectively, to —C(O)-alkyl and —O—C(O)-alkyl groups, where in someembodiments the alkanoyl or alkanoyloxy groups each contain 2-5 carbonatoms. Similarly, the terms “aryloyl” and “aryloyloxy” respectivelyrefer to —C(O)-aryl and —O—C(O)-aryl groups.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, asubstituted or unsubstituted aryl group bonded to an oxygen atom and asubstituted or unsubstituted aralkyl group bonded to the oxygen atom atthe alkyl. Examples include but are not limited to phenoxy, naphthyloxy,and benzyloxy. Representative substituted aryloxy and arylalkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The term “carboxylic acid” as used herein refers to a compound with a—C(O)OH group. The term “carboxylate” as used herein refers to a —C(O)O—group. A “protected carboxylate” refers to a —C(O)O-G where G is acarboxylate protecting group. Carboxylate protecting groups are wellknown to one of ordinary skill in the art. An extensive list ofprotecting groups for the carboxylate group functionality may be foundin Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G.M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can beadded or removed using the procedures set forth therein and which ishereby incorporated by reference in its entirety and for any and allpurposes as if fully set forth herein.

The term “ester” as used herein refers to —COOR⁷⁰ groups. R⁷⁰ is asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.

The term “amide” (or “amido”) includes C- and N-amide groups, i.e.,—C(O)NR⁷¹R⁷², and —NR⁷¹C(O)R⁷² groups, respectively. R⁷¹ and R⁷² areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl orheterocyclyl group as defined herein. Amido groups therefore include butare not limited to carbamoyl groups (—C(O)NH₂) and formamide groups(—NHC(O)H). In some embodiments, the amide is —NR⁷¹C(O)—(C₁₋₅ alkyl) andthe group is termed “carbonylamino,” and in others the amide is—NHC(O)-alkyl and the group is termed “alkanoylamino.”

The term “nitrile” or “cyano” as used herein refers to the —CN group.

Urethane groups include N- and O-urethane groups, i.e., —NR⁷³C(O)OR⁷⁴and —OC(O)NR⁷³R⁷⁴ groups, respectively. R⁷³ and R⁷⁴ are independently asubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R⁷³may also be H.

The term “amine” (or “amino”) as used herein refers to —NR⁷⁵R⁷⁶ groups,wherein R⁷⁵ and R⁷⁶ are independently hydrogen, or a substituted orunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,heterocyclylalkyl or heterocyclyl group as defined herein. In someembodiments, the amine is alkylamino, dialkylamino, arylamino, oralkylarylamino. In other embodiments, the amine is NH₂, methylamino,dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino,phenylamino, or benzylamino.

The term “sulfonamido” includes S- and N-sulfonamide groups, i.e.,—SO₂NR⁷⁸R⁷⁹ and —NR⁷⁸SO₂R⁷⁹ groups, respectively. R⁷⁸ and R⁷⁹ areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, orheterocyclyl group as defined herein. Sulfonamido groups thereforeinclude but are not limited to sulfamoyl groups (—SO₂NH₂). In someembodiments herein, the sulfonamido is —NHSO₂-alkyl and is referred toas the “alkylsulfonylamino” group.

The term “thiol” refers to —SH groups, while sulfides include —SR⁸⁰groups, sulfoxides include —S(O)R⁸¹ groups, sulfones include —SO₂R⁸²groups, and sulfonyls include —SO₂OR⁸³. R⁸⁰, R⁸¹, R², and R⁸³ are eachindependently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl,alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group asdefined herein. In some embodiments the sulfide is an alkylthio group,—S-alkyl.

The term “urea” refers to —NR⁸⁴—C(O)—NR⁸⁵R⁸⁶ groups. R⁸⁴, R⁸⁵, and R⁸⁶groups are independently hydrogen, or a substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, orheterocyclylalkyl group as defined herein.

The term “amidine” refers to —C(NR⁸⁷)NR⁸⁸R⁸⁹ and —NR⁸⁷C(NR⁸⁸)R⁸⁹,wherein R⁸⁷, R⁸⁸, and R⁸⁹ are each independently hydrogen, or asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The term “guanidine” refers to —NR⁹⁰C(NR⁹¹)NR⁹²R⁹³, wherein R⁹⁰, R⁹¹,R⁹² and R⁹³ are each independently hydrogen, or a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein.

The term “enamine” refers to —C(R⁹⁴)═C(R⁹⁵)NR⁹⁶R⁹⁷ and—NR⁹⁴C(R⁹⁵)═C(R⁹⁶)R⁹⁷, wherein R⁹⁴, R⁹⁵, R⁹⁶ and R⁹⁷ are eachindependently hydrogen, a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “halogen” or “halo” as used herein refers to bromine, chlorine,fluorine, or iodine. In some embodiments, the halogen is fluorine. Inother embodiments, the halogen is chlorine or bromine.

The term “hydroxyl” as used herein can refer to —OH or its ionized form,—O—.

The term “imide” refers to —C(O)NR⁹⁸C(O)R⁹⁹, wherein R⁹⁸ and R⁹⁹ areeach independently hydrogen, or a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “imine” refers to —CR¹⁰⁰ (NR¹⁰¹) and —N(CR¹⁰⁰R¹⁰¹) groups,wherein R¹⁰⁰ and R¹⁰¹ are each independently hydrogen or a substitutedor unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein, with theproviso that R¹⁰⁰ and R¹⁰¹ are not both simultaneously hydrogen.

The term “nitro” as used herein refers to an —NO₂ group.

The term “trifluoromethyl” as used herein refers to —CF₃.

The term “trifluoromethoxy” as used herein refers to —OCF₃.

The term “azido” refers to —N₃.

The term “trialkyl ammonium” refers to a —N(alkyl)₃ group. Atrialkylammonium group is positively charged and thus typically has anassociated anion, such as halogen anion.

The term “trifluoromethyldiazirido” refers to

The term “isocyano” refers to —NC.

The term “isothiocyano” refers to —NCS.

The term “pentafluorosulfanyl” refers to —SF₅.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 atoms refers to groupshaving 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers togroups having 1, 2, 3, 4, or 5 atoms, and so forth.

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the present technology and include acid or baseaddition salts which retain the desired pharmacological activity and isnot biologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When the compound ofthe present technology has a basic group, such as, for example, an aminogroup, pharmaceutically acceptable salts can be formed with inorganicacids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuricacid, and phosphoric acid), organic acids (e.g., alginate, formic acid,acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid,tartaric acid, lactic acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (suchas aspartic acid and glutamic acid). When the compound of the presenttechnology has an acidic group, such as for example, a carboxylic acidgroup, it can form salts with metals, such as alkali and earth alkalimetals (e.g., Na⁺, Li⁺, K+, Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines(e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, triethanolamine) or basic aminoacids (e.g., arginine, lysine and ornithine). Such salts can be preparedin situ during isolation and purification of the compounds or byseparately reacting the purified compound in its free base or free acidform with a suitable acid or base, respectively, and isolating the saltthus formed.

Those of skill in the art will appreciate that compounds of the presenttechnology may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or stereoisomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, stereochemical orgeometric isomeric forms, it should be understood that the presenttechnology encompasses any tautomeric, conformational isomeric,stereochemical and/or geometric isomeric forms of the compounds havingone or more of the utilities described herein, as well as mixtures ofthese various different forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The presence and concentrations of theisomeric forms will depend on the environment the compound is found inand may be different depending upon, for example, whether the compoundis a solid or is in an organic or aqueous solution. For example, inaqueous solution, quinazolinones may exhibit the following isomericforms, which are referred to as tautomers of each other:

As another example, guanidines may exhibit the following isomeric formsin protic organic solution, also referred to as tautomers of each other:

Because of the limits of representing compounds by structural formulas,it is to be understood that all chemical formulas of the compoundsdescribed herein represent all tautomeric forms of compounds and arewithin the scope of the present technology.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these stereoisomersare all within the scope of the present technology.

The compounds of the present technology may exist as solvates,especially hydrates. Hydrates may form during manufacture of thecompounds or compositions comprising the compounds, or hydrates may formover time due to the hygroscopic nature of the compounds. Compounds ofthe present technology may exist as organic solvates as well, includingDMF, ether, and alcohol solvates among others. The identification andpreparation of any particular solvate is within the skill of theordinary artisan of synthetic organic or medicinal chemistry.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Alsowithin this disclosure are Arabic numerals referring to referencedcitations, the full bibliographic details of which are provided insections within the Examples. The disclosures of these publications,patents and published patent specifications are hereby incorporated byreference into the present disclosure to more fully describe the presenttechnology.

The Present Technology

In general, there is a need for radiotherapeutic compounds thataccumulate to a greater degree in tumors without unacceptable uptake innormal non-target tissues and organs, as absorbed dose is a function ofthe integral of cumulative activity. By targeting a cellular marker on aneoplastic tissue, the radiotherapeutic can be delivered to the tumorpreferentially, thereby decreasing uptake in non-target tissues.Furthermore, by including additional targeting structures on thecompound, the pharmacokinetics of the compound can be altered. Forexample, using a blood-targeting moiety can increase circulatoryresidence time, which has the effects of increasing tumor perfusion andloading while reducing accumulation of the radiotherapy compound innon-target tissues. Additionally, although targeted radiotherapy hasbeen practiced for some time using macrocyclic complexes ofradionuclides, the macrocycles currently in use (e.g., DOTA) generallyform complexes with many radionuclide metals, such as actinium, radium,bismuth, astatine, lutetium, and lead isotopes among others.Alpha-emitting radionuclides can provide greaterer cytotoxic effects,and thus for therapy are considered substantially more potent, thanbeta-emitting radionuclides. The instability of currently knownmacrocyclic-containing compounds can result in some dissociation of theradionuclide from the macrocycle, and this results in a lack ofselectivity to targeted tissue, which also results in toxicity tonon-targeted tissue. Thus, in addition to the multiple targetingdomains, novel macrocyclic-containing compounds can be incorporated,that display increased retention of the chelated metal.

The present technology provides new compounds that overcome the problemsof traditional radiotherapy compounds, particularly important when usingalpha-emitters, accumulating the compound to a greater degree in tumorswithout unacceptable uptake in normal tissues and organs. The presenttechnology also includes macrocyclic complexes that are substantiallymore stable than those of the conventional art, providing for the use ofalpha-emitting radionuclides instead of beta radionuclides. Thus, thecompouds of the present technology advantageously target cancer cellsmore effectively, with substantially less toxicity to non-targetedtissue than complexes of the art. Moreover, the new complexes canadvantageously be produced at room temperature, in contrast to DOTA-typecomplexes, which generally require elevated temperatures (e.g., at least80° C.) for complexation with the radionuclide.

Thus, in one aspect of the present technology, a compound is providedthat includes a tumor-binding domain, an albumin-binding domain, and acytocidal or cytostatic therapeutic agent, where the tumor-bindingdomain includes an active site that is distal to and stericallyunimpeded by the albumin-binding domain and the therapeutic agent, andthe relative affinity of the tumor-binding domain and thealbumin-binding domain differ in specific affinity by a factor of atleast 100 to about 10,000—such as 100, about 500, 1,000, about 1,500,about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about7,000, about 8,000, about 9,000, about 10,000, or any range includingand/or in between any two of these values. The term “stericallyunimpeded,” as used herein, refers to non-interference between therespective domains of the compound, i.e., the tumor-binding domain andthe albumin-binding domain of the compound each having sufficientflexibility and length such that each can bind to their cognate target,and where the respective domains are spaced apart sufficiently such thatone domain does not physically occlude the binding site of the other, orcause electrostatic effects that significantly reduce the bindingaffinity of the other domain for its' target. The term will bewell-appreciated by those in the biology and chemistry fields, such asevidenced by Rong et al., Molecular modeling of the interactions ofglutamate carboxypeptidase II with its potent NAAG-based inhibitors, JMed Chem. 2002 Sep. 12; 45(19):4140-52, which models the active site ofPSMA and details numerous compounds having affinity for it; see alsoKim. J. K. et al. Synthesis and Properties of a Sterically Unencumbered6-Silanediol Amino Acid. J. Org. Chem. 2012, 77, 2901-2906; Feng, L. etal. An Extremely Facile Aza-Bergman Rearrangement of StericallyUnencumbered Acyclic 3-Aza-3-ene-1,5-diynes. J. Org. Chem. 2003, 68,2234-2242; and Friedrichsen, B. P. et al. Sterically EncumberedFunctional Groups: An Investigation of Endo versus Exo PhosphorylComplexation Using Proton and Phosphorus-31 NMR. J. Am. Chem. Soc. 1990,112, 8931-8941.

In a related aspect, a compound is provided that is a multi-targetedagent having a plurality of sterically unimpeded targeting domains,where the compound includes

-   -   a first targeting domain that includes a blood-protein binding        domain having specific affinity for binding human serum albumin        in the range of about 0.25 to 50 micromolar, and    -   a second targeting domain that includes a tumor-binding domain        having specific affinity for a tumor associated molecular target        in the range of about 0.1 to 75 nanomolar; and    -   a therapeutic domain that includes a cytocidal or cytostatic        therapeutic agent;        wherein the relative affinities of the first and second        targeting domains differ in specific affinity by a factor of at        least 100 to about 10,000—such as 100, about 500, 1,000, about        1,500, about 2,000, about 3,000, about 4,000, about 5,000, about        6,000, about 7,000, about 8,000, about 9,000, about 10,000, or        any range including and/or in between any two of these values.

The first targeting domain that includes the blood-protein bindingdomain may have a specific affinity for binding human serum albumin ofabout 0.25 micromolar, about 0.30 micromolar, about 0.35 micromolar,about 0.40 micromolar, about 0.45 micromolar, about 0.50 micromolar,about 0.55 micromolar, about 0.60 micromolar, about 0.65 micromolar,about 0.70 micromolar, about 0.75 micromolar, about 0.80 micromolar,about 0.85 micromolar, about 0.90 micromolar, about 0.95 micromolar,about 1 micromolar, about 2 micromolar, about 3 micromolar, about 4micromolar, about 5 micromolar, about 10 micromolar, about 15micromolar, about 20 micromolar, about 25 micromolar, about 30micromolar, about 35 micromolar, about 40 micromolar, about 45micromolar, about 50 micromolar, or any range including and/or inbetween any two of these values. The second targeting domain thatincludes the tumor-binding domain may have a specific affinity for thetumor associated molecular target of about 0.1 nanomolar, about 0.15nanomolar, about 0.20 nanomolar, about 0.25 nanomolar, about 0.30nanomolar, about 0.35 nanomolar, about 0.40 nanomolar, about 0.45nanomolar, about 0.50 nanomolar, about 0.55 nanomolar, about 0.60nanomolar, about 0.65 nanomolar, about 0.70 nanomolar, about 0.75nanomolar, about 0.80 nanomolar, about 0.85 nanomolar, about 0.90nanomolar, about 0.95 nanomolar, about 1 nanomolar, about 2 nanomolar,about 3 nanomolar, about 4 nanomolar, about 5 nanomolar, about 10nanomolar, about 15 nanomolar, about 20 nanomolar, about 25 nanomolar,about 30 nanomolar, about 35 nanomolar, about 40 nanomolar, about 45nanomolar, about 50 nanomolar, about 55 nanomolar, about 60 nanomolar,about 65 nanomolar, about 70 nanomolar, about 75 nanomolar, or any rangeincluding and/or in between any two of these values.

In a further related aspect, a compound is provided that is a trefoilconstruct, where the trefoil construct includes a tumor recognitionmoiety, an albumin binding moiety, and a radionuclide moiety that doesnot participate strongly in either tumor target recognition or albuminbinding.

The tumor-binding domain (e.g., the tumor recognition moiety) includes amoiety capable of recognizing or interacting with a molecular target onthe surface of tumor cells. Such molecular targets include cell surfaceproteins such as receptors, enzymes, and antigens. For example, themolecular target may be a receptor, an enzyme, and/or an antigenexpressed on a tumor cell surface (such as a tumor-specific cell surfaceprotein) capable of interacting with the tumor-binding domain. Anexample of such a tumor-binding domain is the glutamate-urea-lysinemotif recognized by prostate specific membrane antigen (PSMA) which isexpressed on the surface of most prostate cancer cells. Another exampleis edotreotide, recognized by somatostatin receptors expressed on thesurface of many neuroendocrine cancers. Thus, the tumor-binding domain(e.g., the tumor recognition moiety) of any aspect and embodiment hereinis capable of binding to a tumor associated molecular target thatincludes one or more of: a tumor associated molecular target that is atumor-specific cell surface protein or other marker such as prostatespecific membrane antigen (PSMA), somatostatin peptide receptor-2(SSTR2), alphavbeta3 (αvβ3), alphavbeta6, a gastrin-releasing peptidereceptor, a seprase, fibroblast activation protein alpha (FAP-alpha), anincretin receptor, a glucose-dependent insulinotropic polypeptidereceptor, VIP-1, NPY, a folate receptor, LHRH, a neuronal transporter(e.g., noradrenaline transporter (NET)), EGFR, HER-2, VGFR, MUC-1, CEA,MUC-4, ED2,TF-antigen, an endothelial specific marker, neuropeptide Y,uPAR, TAG-72, a claudin, a CCK analog, VIP, bombesin, VEGFR, atumor-specific cell surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, acaspace, cMET, or an overexpressed peptide receptor. The preceeding aresimply representative tumor associated molecular targets and for whichdetailed structural information exists for both the target and compoundsthat bind it. The various antibodies, peptides and compounds thatdisplay specific affinity for these particular cellular targets arewidely described in the scientific literature, and can be adapted to theinstant invention as tumor-binding domains. The tumor-binding domain(e.g., the tumor recognition moiety) of any aspect and embodiment hereinis capable of binding to the tumor associated molecular target with atleast moderate adffinity, to high affinity (e.g., the equilibriumbinding constant (K_(D)) ranging from about 10{circumflex over ( )}-8Mto about 10{circumflex over ( )}-10M). Currently preferred tumor-bindingthat target and bind to the active site of PSMA, include for example, aglutamate-ureido-amino acid sequence, a glutamate-urea-lysine sequencewith or without an aromatic substituent at the epsilon amine of lysine,or any derivative thereof that can bind the active site of PSMA withmoderate to high affinity. Exemplary structures are provided herein,however other regions of PSMA can be targeted, and these areinterchangeable with the PSMA tumor-binding domains in the compoundsdetailed herein.

The blood-protein binding domain (e.g., the albumin-binding domain; thealbumin-binding moiety) plays a role in modulating the rate of bloodplasma clearance of the compounds in a subject, thereby increasingcirculation time and compartmentalizing the cytotoxic action ofcytotoxin-containing domain and/or imaging capability of the imagingagent-containing domain in the plasma space instead of normal organs andtissues that may express antigen. Without being bound by theory, thiscomponent of the compound is believed to interact reversibly with serumproteins, such as albumin and/or cellular elements. The affinity of thisblood-protein binding domain (e.g., the albumin-binding domain; thealbumin-binding moiety) for plasma or cellular components of the bloodmay be configured to affect the residence time of the compounds in theblood pool of a subject. In any embodiment herein, the blood-proteinbinding domain (e.g., the albumin-binding domain; the albumin-bindingmoiety) may be configured so that it binds reversibly or non-reversiblywith albumin when in blood plasma.

By way of example, the blood-protein binding domain of any aspect orembodiment herein may include a short-chain fatty acid, medium-chainchain fatty acid, a long-chain fatty acid, myristic acid, a substitutedor unsubstituted indole-2-carboxylic acid, a substituted orunsubstituted thioamide, a substituted or unsubstituted4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid, a substitutedor unsubstituted naphthalene acylsulfonamide, a substituted orunsubstituted diphenylcyclohexanol phosphate ester, a substituted orunsubstituted 4-iodophenylalkanoic acid, a substituted or unsubstituted3-(4-iodophenyl)propionic acid, a substituted or unsubstituted2-(4-iodophenyl)acetic acid, or a substituted or unsubstituted4-(4-iodophenyl)butanoic acid. Certain representative examples ofmoieties that bind the blood protein albumin, that may be included inany embodiment herein include one or more of the following:

The cytocidal or cytostatic therapeutic agent of any aspect andembodiment herein may include a toxin, a venom, a metabolic poison, achemotherapeutic agent, an auger electron-emitting radionuclide, abeta-emitting radionuclide, or an alpha-emitting radionuclide. A moietythat includes a radionuclide is also referred to herein as a“radionuclide moiety.” The cytocidal or cytostatic therapeutic agent ofany aspect and embodiment herein may include a covalently conjugatedchelating agent or a polyaza polycarboxylic macrocycle (collectively,“chelators”) which may further chelate a metal ion; the radionuclidemoiety may include a a covalently conjugated chelating agent or apolyaza polycarboxylic macrocycle chelating a radionuclide. Suchchelated metal ions may provide that the compounds of the presenttechnology may be used in, e.g., magnetic resonance imaging,luminescence imaging, radiotherapy, or a combination of any two or morethereof. The metal ion of any aspect and embodiment herein may be aradionuclide, such as ¹⁷⁷Lu³⁺, ¹⁷⁵Lu³⁺, ⁴⁵Sc³⁺, ⁶⁶Ga³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺,⁶⁹Ga³⁺, ⁷¹Ga³⁺, ⁹⁰Y³⁺, ⁸⁹Y³⁺, ⁸⁶Y³⁺, ⁸⁹Zr⁴⁺, ⁹⁰Y³⁺, ^(99m)Tc⁺¹, ¹¹¹In³⁺,¹¹³In³⁺, ¹¹⁵In³⁺, ¹³⁹La³⁺, ¹³⁶Ce³⁺, ¹³⁸Ce³⁺, ¹⁴⁰Ce³⁺, ¹⁴²Ce³⁺, ¹⁵¹Eu³⁺,¹⁵³Eu³⁺, ¹⁵²Dy³⁺, ¹⁴⁹Tb³⁺, ¹⁵⁹Tb³⁺, ¹⁵⁴Gd³⁺, ¹⁵⁵Gd³⁺, ¹⁵⁶Gd³⁺, ¹⁵⁷Gd³⁺,¹⁵⁸Gd³⁺, ¹⁶⁰Gd³⁺, ¹⁸⁸Re⁺¹, ¹⁸⁶Re⁺¹, ²¹³Bi³⁺, ²¹¹At⁺, ²¹⁷At⁺, ²²⁷Th⁴⁺,²²⁶Th⁴⁺, ²²⁵Ac³⁺, ²³³Ra²⁺, ¹⁵²Dy³⁺, ²¹³Bi³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹²Pb²⁺,²¹²Pb⁴⁺, ²⁵⁵Fm³⁺, or uranium-230. For example, the the metal ion may bean alpha-emitting radionuclide such as ²¹³Bi³⁺, ²¹¹At⁺, ²²⁵Ac³⁺,¹⁵²Dy³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹⁷At⁺, ²²⁷Th⁴⁺, ²²⁶Th⁴⁺, ²³³Ra²⁺, ²¹²Pb²⁺,and ²¹²Pb⁴⁺; for example, the the metal ion may be a beta-emittingradionuclide such as ¹⁷⁷Lu³⁺, ⁹⁰Y³⁺, ¹⁸⁸Re⁺¹, and ¹⁸⁶Re⁺¹. Theradionuclide of any embodiment herein may be a therapeutic radionuclide,a diagnostic radionuclide, or both.

Chelating agents and polyaza polycarboxylic macrocycles useful in anyembodiment of the present technology include, but are not limited to, acovalently conjugated substituted or unsubstituted member of thefollowing group:

-   -   1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),    -   1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),    -   p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS),    -   PIP-DOTA,    -   diethylenetriaminepentaacetic acid (DTPA),    -   PIP-DTPA,    -   AZEP-DTPA,    -   ethylenediamine tetraacetic acid (EDTA),    -   triethylenetetraamine-N,N,N′,N″,N″′,N″′-hexa-acetic acid (TTHA),    -   7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic        acid (DEPA),    -   2,2′,2″-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)        pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic        acid (3p-C-DEPA-NCS),    -   NETA,    -   {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perbydro-1,4,7-triazonin-1-yl}-acetic        acid (NPTA),    -   diacetylpyridinebis(benzoylhydrazone),    -   1,4,7,10,13,16-hexaazacyclooctadecane-N,N′,N″,N″′,N″″,N″″′-hexaaceticacid        (HEHA),    -   octadentate terephthalamide ligands,    -   siderophores,    -   2,2′-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic        acid,    -   N,N-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6        (H2macropa),    -   6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic        acid (macropa-NCS), and    -   3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene.        Certain members of this exemplary group are illustrated below.

It is to be understood that a “covalently conjugated” chelating agent ora “covalently conjugated” polyaza polycarboxylic macrocycle means achelating agent or polyaza polycarboxylic macrocycle (such as thoselisted above) wherein one or more bonds to a hydrogen atom containedtherein are replaced by a bond to an atom of the remainder of thecompound of the present technology or a pi bond between two atoms isreplaced by a bond from one of the two atoms to the compound of thepresent technology and the other of the two atoms includes a new bond,e.g. to a hydrogen (such as reaction of an —NCS group in a chelatingagent to provide the covalently conjugated chelating agent).

The present technology also provides compositions and medicamentscomprising anyone of the aspects and embodiments of the compounds of thepresent technology and a pharmaceutically acceptable carrier or one ormore excipients or fillers (collectively refered to as “pharmaceuticallyacceptable carrier” unless otherwise specified). The compositions may beused in the methods and treatments described herein. The presenttechnology also provides pharmaceutical compositions including apharmaceutically acceptable carrier and an effective amount of acompound of any one of the aspects and embodiments of the compounds ofthe present technology for imaging and/or treating a condition; andwhere the condition may include a glioma, a breast cancer, an adrenalcortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and/or aprostate cancer. For example, such conditions may include a mammaliantissue overexpressing PSMA, such as a cancer expressing PSMA (includingcancer tissues, cancer related neo-vasculature, or a combinationthereof), Crohn's disease, or IBD.

In a further related aspect, an imaging method is provided that includesadministering a compound of any one of the aspects and embodiments ofthe compounds of the present technology (e.g., such as administering aneffective amount) or administering a pharmaceutical compositioncomprising an effective amount of a compound of any one of the aspectsand embodiments of the compounds of the present technology to a subjectand, subsequent to the administering, detecting positron emission,detecting gamma rays from positron emission and annihilation (such as bypositron emission tomography), and/or detecting Cerenkov radiation dueto positron emission (such as by Cerenkov luminescene imaging). In anyembodiment of the imaging method, the subject may be suspected ofsuffering from a condition that includes a glioma, a breast cancer, anadrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, a prostatecancer, a mammalian tissue overexpressing PSMA, such as a cancerexpressing PSMA (including cancer tissues, cancer relatedneo-vasculature, or a combination thereof), Crohn's disease, or IBD. Thedetecting step may occur during a surgical procedure on a subject, e.g.,to remove a mammalian tissue overexpressing PSMA. The detecting step mayinclude use of a handheld device to perform the detecting step. Forexample, Cerenkov luminescene images may be acquired by detecting theCerenkov light using ultra-high-sensitivity optical cameras such aselectron-multiplying charge-coupled device (EMCCD) cameras.

In any of the above embodiments, the effective amount may be determinedin relation to a subject. “Effective amount” refers to the amount of acompound or composition required to produce a desired effect. Onenon-limiting example of an effective amount includes amounts or dosagesthat yield acceptable toxicity and bioavailability levels fortherapeutic (pharmaceutical) use including, but not limited to, thetreatment of e.g., a glioma, a breast cancer, an adrenal corticalcancer, a cervical carcinoma, a vulvar carcinoma, an endometrialcarcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma,a non-small cell lung cancer, a small cell lung cancer, a bladdercancer, a colon cancer, a primary, gastric adenocarcinoma, a primarycolorectal adenocarcinoma, a renal cell carcinoma, and a prostatecancer. Another example of an effective amount includes amounts ordosages that are capable of reducing symptoms associated with e.g., aglioma, a breast cancer, an adrenal cortical cancer, a cervicalcarcinoma, a vulvar carcinoma, an endometrial carcinoma, a primaryovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lungcancer, a small cell lung cancer, a bladder cancer, a colon cancer, aprimary, gastric adenocarcinoma, a primary colorectal adenocarcinoma, arenal cell carcinoma, or a prostate cancer, such as, for example,reduction in proliferation and/or metastasis. An effective amount of acompound of the present technology may include an amount sufficient toenable detection of binding of the compound to a target of interestincluding, but not limited to, one or more of a glioma, a breast cancer,an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, or a prostatecancer (such as castration resistant prostate cancer). Another exampleof an effective amount includes amounts or dosages that are capable ofproviding a detectable gamma ray emission from positron emission andannihilation (above background) in a subject with a tissueoverexpressing PSMA, such as, for example, statistically significantemission above background. Another example of an effective amountincludes amounts or dosages that are capable of providing a detectableCerenkov radiation emission due to positron emission above background)in a subject with a tissue overexpressing PSMA, such as, for example,statistically significant emission above background. The effectiveamount may be from about 0.01 g to about 1 mg of the compound per gramof the composition, and preferably from about 0.1 g to about 500 g ofthe compound per gram of the composition.

As used herein, a “subject” or “patient” is a mammal, such as a cat,dog, rodent or primate. Typically the subject is a human, and,preferably, a human suffering from or suspected of suffering from aglioma, a breast cancer, an adrenal cortical cancer, a cervicalcarcinoma, a vulvar carcinoma, an endometrial carcinoma, a primaryovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lungcancer, a small cell lung cancer, a bladder cancer, a colon cancer, aprimary, gastric adenocarcinoma, a primary colorectal adenocarcinoma, arenal cell carcinoma, or a prostate cancer. The term “subject” and“patient” can be used interchangeably.

In particular, the effective amount of a compound of any embodimentherein for treating a cancer and/or a mammalian tissue overexpressingPSMA may be from about 0.1 μg to about 50 μg per kilogram of the mass ofthe subject. Thus, for treating a cancer (e.g., a glioma, a breastcancer, an adrenal cortical cancer, a cervical carcinoma, a vulvarcarcinoma, an endometrial carcinoma, a primary ovarian carcinoma, ametastatic ovarian carcinoma, a non-small cell lung cancer, a small celllung cancer, a bladder cancer, a colon cancer, a primary, gastricadenocarcinoma, a primary colorectal adenocarcinoma, a renal cellcarcinoma, a prostate cancer, and/or a castration resistant prostatecancer) and/or a mammalian tissue overexpressing PSMA; the effectiveamount of a compound of any embodiment described herein may be about 0.1μg/kg, about 0.2 μg/kg, about 0.3 μg/kg, about 0.4 μg/kg, about 0.5μg/kg, about 0.6 μg/kg, about 0.7 μg/kg, about 0.8 μg/kg, about 0.9μg/kg, about 1 μg/kg, about 2 μg/kg, about 3 μg/kg, about 4 μg/kg, about5 μg/kg, about 6 μg/kg, about 7 μg/kg, about 8 μg/kg, about 9 μg/kg,about 10 μg/kg, about 11 μg/kg, about 12 μg/kg, about 13 μg/kg, about 14μg/kg, about 15 μg/kg, about 16 μg/kg, about 17 μg/kg, about 18 μg/kg,about 19 μg/kg, about 20 μg/kg, about 22 μg/kg, about 24 μg/kg, about 26μg/kg, about 28 μg/kg, about 30 μg/kg, about 32 μg/kg, about 34 μg/kg,about 36 μg/kg, about 38 μg/kg, about 40 μg/kg, about 42 μg/kg, about 44μg/kg, about 46 μg/kg, about 48 μg/kg, about 50 μg/kg, or any rangeincluding and/or in between any two of these values.

In particular, the effective amount of a compound of any embodimentherein for imaging a cancer and/or a mammalian tissue overexpressingPSMA may be from about 0.1 μg to about 50 μg per kilogram of the mass ofthe subject. Thus, for treating a cancer (e.g., a glioma, a breastcancer, an adrenal cortical cancer, a cervical carcinoma, a vulvarcarcinoma, an endometrial carcinoma, a primary ovarian carcinoma, ametastatic ovarian carcinoma, a non-small cell lung cancer, a small celllung cancer, a bladder cancer, a colon cancer, a primary, gastricadenocarcinoma, a primary colorectal adenocarcinoma, a renal cellcarcinoma, a prostate cancer, and/or a castration resistant prostatecancer) and/or a mammalian tissue overexpressing PSMA; the effectiveamount of a compound of any embodiment described herein may be about 0.1μg/kg, about 0.2 μg/kg, about 0.3 μg/kg, about 0.4 μg/kg, about 0.5μg/kg, about 0.6 μg/kg, about 0.7 μg/kg, about 0.8 μg/kg, about 0.9μg/kg, about 1 μg/kg, about 2 μg/kg, about 3 μg/kg, about 4 μg/kg, about5 μg/kg, about 6 μg/kg, about 7 μg/kg, about 8 μg/kg, about 9 μg/kg,about 10 μg/kg, about 11 μg/kg, about 12 μg/kg, about 13 μg/kg, about 14μg/kg, about 15 μg/kg, about 16 μg/kg, about 17 μg/kg, about 18 μg/kg,about 19 μg/kg, about 20 μg/kg, about 22 μg/kg, about 24 μg/kg, about 26μg/kg, about 28 μg/kg, about 30 μg/kg, about 32 μg/kg, about 34 μg/kg,about 36 μg/kg, about 38 μg/kg, about 40 μg/kg, about 42 μg/kg, about 44μg/kg, about 46 μg/kg, about 48 μg/kg, about 50 μg/kg, or any rangeincluding and/or in between any two of these values.

The compounds of the present technology may also be administered to apatient along with other conventional imaging agents that may be usefulin the imaging and/or treatment of a glioma, a breast cancer, an adrenalcortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, a prostatecancer, or a mammalian tissue overexpressing PSMA. Such mammaliantissues include, but are not limited to, a cancer expressing PSMA(including cancer tissues, cancer related neo-vasculature, or acombination thereof), Crohn's disease, or IBD. Thus, a pharmaceuticalcomposition and/or method of the present technology may further includean imaging agent different than the compounds of Formulas I-III; apharmaceutical composition and/or method of the present technology mayinclude an treatment agent different than the compounds of the presenttechnology; a pharmaceutical composition and/or method of the presenttechnology may further include an imaging agent according to anyembodiment of a compound of the present technology and therapeutic agentthat is also according to any embodiment of a compound of the presenttechnology. It may be that the compound according to the presenttechnology is both a therapeutic agent and an imaging agent. Theadministration may include oral administration, parenteraladministration, or nasal administration. In any of these embodiments,the administration may include subcutaneous injections, intravenousinjections, intraperitoneal injections, or intramuscular injections. Inany of these embodiments, the administration may include oraladministration. The methods of the present technology may also includeadministering, either sequentially or in combination with one or morecompounds of the present technology, a conventional imaging agent in anamount that can potentially or synergistically be effective for theimaging of a mammalian tissue overexpressing PSMA.

In any of the embodiments of the present technology described herein,the pharmaceutical composition may be packaged in unit dosage form. Theunit dosage form is effective in treating a glioma, a breast cancer, anadrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and/or aprostate cancer. Generally, a unit dosage including a compound of thepresent technology will vary depending on patient considerations. Suchconsiderations include, for example, age, protocol, condition, sex,extent of disease, contraindications, concomitant therapies and thelike. An exemplary unit dosage based on these considerations may also beadjusted or modified by a physician skilled in the art. For example, aunit dosage for a patient comprising a compound of the presenttechnology may vary from 1×10 g/kg to 1 g/kg, preferably, 1×10⁻³ g/kg to1.0 g/kg. Dosage of a compound of the present technology may also varyfrom 0.01 mg/kg to 100 mg/kg or, preferably, from 0.1 mg/kg to 10 mg/kg.Suitable unit dosage forms, include, but are not limited to powders,tablets, pills, capsules, lozenges. suppositories. patches. nasalsprays, injectibles, implantable sustained-release formulations,rnucoadherent films, topical varnishes, lipid complexes, etc.

The pharmaceutical compositions may be prepared by mixing one or morecompounds of the present technology with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like to prevent and treatdisorders associated with cancer (e.g., a glioma, a breast cancer, anadrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary, gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and aprostate cancer). The compounds and compositions described herein may beused to prepare formulations and medicaments that treat e.g., a glioma,a breast cancer, an adrenal cortical cancer, a cervical carcinoma, avulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma,a metastatic ovarian carcinoma, a non-small cell lung cancer, a smallcell lung cancer, a bladder cancer, a colon cancer, a primary, gastricadenocarcinoma, a primary colorectal adenocarcinoma, a renal cellcarcinoma, and a prostate cancer. Such compositions may be in the formof, for example, granules, powders, tablets, capsules, syrup,suppositories, injections, emulsions, elixirs, suspensions or solutions.The instant compositions may be formulated for various routes ofadministration, for example, by oral, parenteral, topical, rectal,nasal, vaginal administration, or via implanted reservoir. Parenteral orsystemic administration includes, but is not limited to, subcutaneous,intravenous, intraperitoneal, and intramuscular, injections. Thefollowing dosage forms are given by way of example and should not beconstrued as limiting the instant present technology.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant present technology, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Typically, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Compounds of the present technology may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. The carriers and stabilizers varywith the requirements of the particular compound, but typically includenonionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant)aerosols are typically used for delivery of compounds of the presenttechnology by inhalation.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instant presenttechnology. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference. The instant compositions mayalso include, for example, micelles or liposomes, or some otherencapsulated form.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant present technology.

Various assays and model systems can be readily employed to determinethe therapeutic effectiveness of the treatment according to the presenttechnology.

For the indicated condition, test subjects will exhibit a 10%, 20%, 30%,50% or greater reduction, up to a 75-90%, or 95% or greater, reduction,in one or more symptom(s) caused by, or associated with, the disorder inthe subject, compared to placebo-treated or other suitable controlsubjects.

The present technology further provides a method of achieving an in vivotissue distribution of a radiotherapeutic in a mammalian subject inwhich a ratio of tumor activity to kidney activity of 1 or greater isobserved within about 4 hours to about 24 hours of administration of theradiotherapeutic to the mammalian subject. Such a method includesadministering to the mammalian subject the radiotherapeutic, where theradiotherapeutic comprises a first moiety that targets prostate specificmembrane antigen (“PSMA”), a second moiety that bears a radionuclide,and a third moiety that has an affinity for serum albumin, the firstmoiety being separated from the second moiety by a first covalent linkerand the third moiety being separated from the second moiety by a secondcovalent linker. The separation between the first and second moieties(on the basis of a contiguous atom count associated with the firstcovalent linker) is from about 8 atoms to about 40 atoms, and theseparation between the third moiety and the first and second moieties(on the basis of a contiguous atom count associated with the secondcovalent linker) is from about 10 atoms to about 100 atoms.

The method may include obtaining an image of the mammalian subject about4 hours to about 24 hours after administration of the radiotherapeutic;thus, obtaining an image after administration of the radiotherapeuticmay occur after about 4 hours, about 5 hours, about 6 hours, about 7hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours, or anyrange including and/or in between any two of these values. The ratio oftumor activity to kidney activity of 1 or greater may persist up toabout 24 hours after administration of the radiotherapeutic. In anyembodiment herein of the method, it may be that substantially noradionuclide activity is observed in salivary glands of the mammaliansubject about 24 hours to about 48 hours after administration of theradiotherapeutic. In any embodiment herein of the method, it may be thatthe contiguous atom count associated with the first covalent linkerranges from about 10 atoms to about 30 atoms. In any embodiment hereinof the method, it may be that the contiguous atom count associated withthe second covalent linker ranges from about 15 atoms to about 40 atoms.In any embodiment herein of the method, it may be that theadministration comprises intravenous administration.

The examples herein are provided to illustrate advantages of the presenttechnology and to further assist a person of ordinary skill in the artwith preparing or using the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, prodrugs, or tautomericforms thereof. The examples herein are also presented in order to morefully illustrate the preferred aspects of the present technology. Theexamples should in no way be construed as limiting the scope of thepresent technology, as defined by the appended claims. The examples caninclude or incorporate any of the variations, aspects or embodiments ofthe present technology described above. The variations, aspects orembodiments described above may also further each include or incorporatethe variations of any or all other variations, aspects or embodiments ofthe present technology.

Examples Section 1.1

Materials and Instrumentation. All solvents and reagents, unlessotherwise noted, were purchased from commercial sources and used asreceived without further purification. Solvents noted as “dry” wereobtained following storage over 3 A molecular sieves. Reactions weremonitored by thin-layer chromatography (TLC, Whatman UV254aluminum-backed silica gel). The HPLC system used for analysis andpurification of compounds consisted of a CBM-20A communications busmodule, an LC-20AP (preparative) pump, and an SPD-20AV UV/Vis detectormonitoring at 270 nm (Shimadzu, Japan). Purification was performed withan Epic Polar preparative column, 120 Å, 10 μm, 25 cm×20 mm (ESIndustries, West Berlin, N.J.) at a flow rate of 14 mL/min, unlessotherwise noted. Gradient HPLC methods were employed using a binarymobile phase that contained H₂O (A) and either MeOH (B) or ACN (C). HPLCMethod A: 10% B (0-5 min), 10-100% B (5-25 min). Method B: 10% C (0-5min), 10-100% C (5-25 min). Method C: 10% C (0-5 min), 10-100% C (5-40min). Method D: 10% C (0-5 min), 10-100% C (5-20 min). The solventsystems contained 0.2% trifluoroacetic acid (TFA). NMR spectra wererecorded at ambient temperature on Varian Inova 300 MHz, 400 MHz, 500MHz or 600 MHz spectrometers, or on a Bruker AV III HD 500 MHzspectrometer equipped with a broadband Prodigy cryoprobe. Chemicalshifts are reported in ppm. ¹H and ¹³C NMR spectra were referenced tothe TMS internal standard (0 ppm), to the residual solvent peak, or toan acetonitrile internal standard (2.06 ppm in D₂O spectra). ¹⁹F NMRspectra were referenced to a monofluorobenzene internal standard(−113.15 ppm). The splitting of proton resonances in the reported ¹Hspectra is defined as: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, dt=doublet of triplets, td=triplet of doublets, andbr=broad. IR spectroscopy was performed on a KBr pellet of sample usinga Nicolet Avatar 370 DTGS (ThermoFisher Scientific, Waltham, Mass.).High-resolution mass spectra (HRMS) were recorded on an ExactiveOrbitrap mass spectrometer in positive ESI mode (ThermoFisherScientific, Waltham, Mass.). UV/visible spectra were recorded on a Cary8454 UV-Vis (Agilent Technologies, Santa Clara, Calif.) using 1-cmquartz cuvettes, unless otherwise noted. Elemental analysis (EA) wasperformed by Atlantic Microlab, Inc. (Norcross, Ga.).

Preparation of di-tert-butyl((1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)glutamate(5)

(S)-2-[(Imidazole-1-carbonyl)amino]pentanedioic acid di-tertbutyl Ester(2): To a suspension of L-di-tert-butyl glutamate hydrochloride (15.0 g,51 mmol) in DCM (150 mL) cooled to 0° C. was added TEA (18 mL) and DMAP(250 mg). After the mixture was stirred for 5 min, CDI (9.0 g, 56 mmol)was added and the mixture was stirred overnight with warming to roomtemperature. The mixture was diluted with DCM (150 mL) and washed withsaturated sodium bicarbonate (60 mL), water (2×100 mL), and brine (100mL). The organic layer was dried over sodium sulfate and concentrated toafford the crude product as a semi-solid, which slowly solidified uponstanding. The crude material was triturated with hexane/ethyl acetate toafford a white solid which was filtered, washed with hexane (100 mL),and dried to afford 2 (15.9 g, 45 mmol, 88%) as a white solid.

(S)-2-[3((S)-(5-Benzyloxycarbonylamino)-1-tert-butoxycarbonylpentylureido]pentanedioicacid di-tert-butyl Ester (3): To a solution of 2 (1 g, 2.82 mmol) in DCE(10 mL) at 0° C. was added MeOTf (0.47 g, 2.85 mmol) and TEA (0.57 g,5.65 mmol). After the solution was stirred for 30 min, Cbz-L-Lys-Ot-Bu(1.06 g, 2.82 mmol) was added in one portion and allowed to stir for 1 hat 40° C. The mixture was concentrated to dryness and purified by columnchromatography (SiO₂) to afford 3 as a white solid (1.37 g, 79%).

2-[3-(5-Amino-1-tert-butoxycarbonylpentyl)ureido]pentanedioic aciddi-tert-butyl ester (4): To a solution of 3 (630 mg, 1.0 mmol) inethanol (20 mL) under a hydrogen atmosphere was added ammonium formate(630 mg, 10 eq) followed by 10% Pd—C. The suspension was allowed tostand with occasional agitation overnight until complete. The mixturewas filtered through Celite and concentrated to afford the desiredproduct (479 mg, 98%) as a waxy solid.

Di-tert-butyl((1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)glutamate(5): To a solution of 4 (0.488, 1 mmol) in DCM (10 mL) was added1-ethynyl-3-isocyanatobenzene (185 mg, 1.3 mmol) in DCM (5 mL) at r.tunder N₂. The resulting reaction mixture was stirred for 12 h at thesame temperature and transferred to a separating funnel and washed withwater (2×50 mL) and brine (30 mL). The organic layer was dried oversodium sulfate and concentrated to afford the crude product as asemisolid which was purified by column chromatography (SiO₂) to afford 5as a white foam (84%).

Preparation of tert-butyl-N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycyl-N6-(tert-butoxycarbonyl)lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(10)

2,5-dioxopyrrolidin-1-ylN2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysinate(7):N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine6 (4.68 g, 10 mmol) was dissolved in dry DCM (20 mL) and DIPEA (1.74 mL,10 mmol) was added. The reaction mixture was stirred at r.t. for 10 minand solid di(N-succinimidyl) carbonate (3.84 g, 15 mmol) was added inone portion. The resulting reaction mixture was stirred for 3-4 h anddiluted with DCM, transferred to a separating funnel and washed with anexcess of water. The organic layer was collected, dried on MgSO₄ andevaporated to dryness to afford a semi-solid. The crude product wasrecrystallized from ethanol and diethyl ether to give 7 as a creamcolored solid (3.44 g, 61%).

tert-butylN2-(N2-(((9H-fluoren-9-yl)oxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(8): To a suspension of H-Lys(Z)-OtBu HCl (3.72 g, 10 mmol) in DCM (25mL) was added DIPEA (1.74 mL, 10 mmol) at 0° C. followed by dropwiseaddition of compound 7 (5.65 g, 10 mmol) in DCM (20 mL). The resultingclear solution was stirred overnight at r.t. The solvent was evaporatedand the crude compound was purified by column chromatography (SiO₂) toafford 8 as a white solid (76%).

tert-butylN6-((benzyloxy)carbonyl)-N2-(N6-(tert-butoxycarbonyl)-L-lysyl)-L-lysinate(9): To a solution of compound 8 (1.156 g, 2 mmol) in DCM was addeddiethylamine (6 mL) dropwise and the resulting reaction mixture wasstirred at r.t for 4-5 h. Solvents were evaporated under reducedpressure and the crude product was re-dissolved in DCM and washed withwater (2×100 mL), and brine (100 mL). The organic layer was dried oversodium sulfate and concentrated to afford the crude product as asemisolid, which was used as such without any further purification.

tert-butyl-N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycyl-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(10): To a solid mixture of(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycine (246 mg, 0.6mmol) and HATU (230 mg, 0.6 mmol) under N₂ was added dry DMF, and themixture was stirred for 5 min at r.t. DIPEA (0.12 mL, 0.7 mmol) wasadded to the reaction mixture and stirring was continued for 10 min atr.t. A solution of compound 9 (282 mg, 0.5 mmol) in DMF was addeddropwise at r.t and stirred at the same temperature for 12 h. DMF wasevaporated under reduced pressure to give a suspension, which wasdissolved in DCM (10 mL), transferred to a separating funnel and washedwith water (2×20 mL) and brine (15 mL). The organic layer was collected,dried on MgSO₄ and evaporated to dryness to afford a semi-solid. Thecrude compound was purified by column chromatography (SiO₂) to affordthe desired product 10 as a brown solid (41%).

tert-butylN2-(N2-(2-azidoacetyl)glycylglycylglycyl-N6-(tert-butoxycarbonyl)lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate (12)

tert-butylN6-((benzyloxy)carbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-glycylglycylglycyl-L-lysyl)-L-lysinate(11): To a solution of compound 10 (0.478 g, 0.5 mmol) in DCM (10 mL)was added diethylamine (2 mL) dropwise and the resulting reactionmixture was stirred at r.t for 3 h. Solvents were evaporated underreduced pressure and re-dissolved in DCM and washed with water (2×20 mL)and brine (20 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product 11 as a semisolid, which wasused without any further purification.

tert-butylN2-(N2-(2-azidoacetyl)glycylglycylglycyl-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(12): To a solid mixture of azidoacetic acid (101 mg, 1 mmol) and HATU(383 mg, 1 mmol) under N₂ was added dry DMF (5 mL), and the mixture wasstirred for 5 min at r.t. DIPEA (0.17 mL, 1 mmol) was added to thereaction mixture and stirring was continued for 10 min at r.t. Asolution of compound 11 (367 mg, 0.5 mmol) in DMF (5 mL) was addeddropwise at r.t and stirred at the same temperature for 12 h. DMF wasevaporated under reduced pressure to give a suspension, which wasdissolved in DCM (10 mL) and washed with water (2×20 mL), and brine (15mL). The crude compound was used without any further purification.

Preparation of10-(6-alkamido-1-(tert-butoxy)-1-oxohexan-2-yl)-24,28,30-tri-tert-butyl-2,2-dimethyl-4,12,21,26-tetraoxo-3-oxa-5,11,20,25,27-pentaazatriacontane-10,24,28,30-tetracarboxylate(14)

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14,17,20,23-hexaoxo-1-phenyl-2-oxa-4,10,13,16,19,22-hexaazatetracosan-24-yl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(13): Compound 12 (140 mg, 0.1 mmol) and compound 5 (63 mg, 0.1 mmol)were dissolved in DMF (2 mL) and aqueous solutions of 0.5M CuSO₄ and0.5M sodium ascorbate were added subsequently. The resulting reactionwas stirred for 3 h at r.t. DMF was evaporated and the crude compound 13was used without any further purification.

10-(6-alkamido-1-(tert-butoxy)-1-oxohexan-2-yl)-24,28,30-tri-tert-butyl-2,2-dimethyl-4,12,21,26-tetraoxo-3-oxa-5,11,20,25,27-pentaazatriacontane-10,24,28,30-tetracarboxylate(14): Compound 13 (144 mg, 0.1 mmol) was dissolved in a mixture ofmethanol:THF (1:1, 10 mL) and 10% Pd—C was added. The resultingsuspension was stirred under H2 (balloon pressure) atmosphere for 3 h.The mixture was filtered through Celite and concentrated to afford thecorresponding amine (not shown) as semi solid, which was usedimmediately to the next step. To a solid mixture of acid RCOOH (0.1mmol) and HATU (38 mg, 0.1 mmol) under N₂ was added dry DMF (3 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of amine (0.1 mmol) in DMF (2 mL) was addeddropwise at r.t and stirred at the same temperature for 12 h. DMF wasevaporated under reduced pressure to give a suspension, which wasdissolved in DCM (5 mL) and washed with water (2×10 mL) and brine (10mL). The organic layer was dried over sodium sulfate and concentrated toafford the crude product which was purified by column chromatography(SiO₂), and product 14 was isolated as a semi-solid.

Preparation of 15

To a solution of compound 14 (1 eq; R=(4-iodophenyl)CH₂—) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixturewas stirred for 3 h at r.t. Completion of the reaction was monitored byTLC. Solvents were removed under reduced pressure and co-distilled withtoluene (2×5 mL). The amine HCl salts formed were dissolved in DMF (1.5mL) and DIPEA (0.5 mmol, 20 eq) was added. The resulting reactionmixture was stirred for 10 min before adding p-NCS-Bn-DOTA (2 eq) anddistilled water (0.5 mL). Stirring was continued for 3 h at r.t. Thereaction mixture was directly subjected to LCMS purification using 0.1%formic acid in ACN and water. The product was collected and lyophilized.¹H NMR (500 MHz, DMSO-d₆): δ 12.25 (bs, 7H), 9.40 (bs, 1H), 8.64-8.62(m, 1H, N—H), 8.54-8.52 (m, 1H, N—H), 8.42 (s, 1H), 8.34-8.31 (m, 1H,N—H), 8.15-8.11 (m, 3H), 8.04-8.02 (m, 1H, N—H), 7.94-7.91 (m, 2H, N—H),7.64-7.63 (m, 3H), 7.37-7.31 (m, 5H), 7.28-7.25 (m, 1H), 7.17-7.16 (m,2H), 7.05-7.04 (m, 3H), 6.34-6.30 (m, 2H), 6.17 (bs, 1H), 5.21 (s, 2H),4.34-4.30 (m, 1H), 4.13-4.04 (m, 4H), 3.84-3.83 (m, 2H), 3.75-3.74 (m,4H), 3.63-3.60 (m, 3H), 3.16-3.13 (m, 4H), 3.12-3.05 (m, 4H), 3.02-2.98(m, 6H), 2.30-2.18 (m, 3H), 1.95-1.88 (m, 1H), 1.71-1.65 (m, 5H),1.58-1.49 (m, 6H), 1.45-1.22 (m, 12H). ³C NMR (500 MHz, DMSO-d₆): δ174.3, 174.0, 173.5, 173.2, 171.4, 169.3, 168.9, 168.7, 168.2, 165.6,157.1, 154.9, 146.1, 140.9, 136.7, 136.1, 131.2, 130.8, 129.0, 122.6,118.4, 117.8, 116.9, 116.0, 113.9, 53.4, 52.1, 51.9, 51.6, 51.4, 41.9,41.8, 41.6, 41.5, 38.2, 31.8, 31.7, 30.3, 29.7, 29.3, 28.5, 28.0, 27.3,22.7, 22.5, 22.4, 17.9, 16.5, 12.3. HRMS calculated for C₇₃H₁₀₂IN₁₉O₂₄S([M+2H]⁺), 1787.6110, found 1787.6048.

Preparation of2-[3-(5-Amino-1-tert-butoxycarbonylpentyl)ureido]pentanedioic Aciddi-tert-butyl Ester (17)

5-benzyl 1-(tert-butyl)(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate(16): To a solution of 2 (1 g, 2.82 mmol) in DCE (10 mL) at 0° C. wasadded MeOTf (0.47 g, 2.85 mmol) and TEA (0.57 g, 5.65 mmol). After thesolution was stirred for 30 min, H-L-Glu(Bzl)-OtBu hydrochloride (0.927g, 2.82 mmol) was added in one portion and allowed to stir for 1 h at40° C. The mixture was concentrated to dryness and purified by columnchromatography (SiO₂) to afford the desired product as a white solid(79%).

(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoicacid (17): To a solution of 16 in ethanol (20 mL) under a hydrogenatmosphere was added ammonium formate (630 mg, 10 eqv) followed by 10%Pd—C, and the suspension was allowed to stand with occasional agitationovernight until complete. The mixture was filtered through Celite andconcentrated to afford 17, the desired product (479 mg, 98%) as a waxysolid.

Preparation of 2,5-dioxopyrrolidin-1-yl8-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)octanoate (19)

8-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)octanoic acid, 18 (1.43 g,3 mmol) was dissolved in anhydrous DCM (20 mL) and DIPEA (0.522 mL, 3mmol) was added. The reaction mixture was stirred at r.t for 10 min andsolid di(N-succinimidyl) carbonate (1.152 g, 4.5 mmol) was added in oneportion. The resulting reaction mixture was stirred for 3 h and dilutedwith DCM and transferred in to a separating funnel and washed withexcess of water. The organic layer was collected, dried on MgSO₄ andevaporated to dryness to afford a semi-solid, which was recrystallizedfrom ethanol and diethyl ether to give the desired product as anoff-white solid (0.932 g, 65.08%).

Preparation of tetra-tert-butyl(3S,7S,21S,24S)-28-amino-21-(4-((tert-butoxycarbonyl)amino)butyl)-5,10,19,22-tetraoxo-4,6,11,20,23-pentaazaoctacosane-1,3,7,24-tetracarboxylate(23)

tert-butylN2-(N2-(8-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)octanoyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(20): Compound 9 (0.551 g, 1 mmol) and compound 19 (0573, 1.2 mmol) weredissolved in DCM (10 mL) and stirred for 12 b at r.t. Progress of thereaction was monitored by TLC. The mixture was concentrated to drynessand purified by column chromatography (SiO₂) to afford the desiredproduct 20 as a brown solid (41%).

tert-butylN2-(N2-(8-aminooctanoyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate(21): To a solution of compound 20 (0.457 g, 0.5 mmol) in DCM (10 mL)was added diethylamine (3 mL) dropwise and the resulting reactionmixture was stirred at r.t for 3 h. Solvents were evaporated underreduced pressure and re-dissolved in DCM and washed with water (2×20 mL)and brine (20 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product 21 as a semisolid, which wasused without any further purification.

tetra-tert-butyl(9S,12S,26S,30S)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14,23,28-pentaoxo-1-phenyl-2-oxa-4,10,13,22,27,29-hexaazadotriacontane-9,26,30,32-tetracarboxylate(22): To a solid mixture of compound 17 (0.114, 0.24 mmol) and HATU(0.092 g, 0.24 mmol) under N₂ was added dry DMF, and the mixture wasstirred for 5 min at r.t. DIPEA (0.041 mL, 0.24 mmol) was added to thereaction mixture and stirring was continued for 10 min at r.t. Asolution of compound 21 (0.141, 0.2 mmol) in DMF was added dropwise atr.t and stirred at the same temperature for 12 h. DMF was evaporatedunder reduced pressure to give a suspension, which was dissolved in DCM(10 mL) and washed with water (2×20 mL), and brine (15 mL). The organiclayer was dried over sodium sulfate and concentrated to afford the crudeproduct which was purified by column chromatography (SiO₂) to afford theproduct 22 as a semi-solid (28%).

tetra-tert-butyl(3S,7S,21S,24S)-28-amino-21-(4-((tert-butoxycarbonyl)amino)butyl)-5,10,19,22-tetraoxo-4,6,11,20,23-pentaazaoctacosane-1,3,7,24-tetracarboxylate(23): Compound 22 (0.1 g, 0.085 mmol) was dissolved in a mixture ofmethanol:THF (1:1, 10 mL) and 10% Pd—C was added. The resultingsuspension was stirred under H2 (balloon pressure) atmosphere for 3 h.The mixture was filtered through Celite and concentrated to afford thedesired product 23 (91%) as a waxy solid.

Preparation of 24 (a-2) and 25 (a-h)

tetra-tert-butyl(2S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-(4-(4-(4-iodophenyl)butanamido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24a): To a solid mixture of 4-(4-iodophenyl)butanoic acid (0.029 g, 0.1mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL) and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24a was isolated as a semi-solid(18%).

33-(4-iodophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25a): definted, but the a solution of compound 24a (0.025 mmol, 1eq) in dioxane (2 mL) was added 4M HCl in dioxane (2 mL). The resultingreaction mixture was stirred for 3 h at r.t. Completion of the reactionwas monitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.050 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water.

tetra-tert-butyl(2S)-2-(4-(4-(4-bromophenyl)butanamido)butyl)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24b): To a solid mixture of 4-(4-bromophenyl)butanoic acid (0.024 g,0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL)and the mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL), and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24b was isolated as a semi-solid(6%).

33-(4-bromophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25b): To a solution of compound 24b (0.020 mmol, 1 eq) in dioxane(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.050 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25b.

tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-(4-(4-(4-iodophenyl)butanamido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24a): To a solid mixture of 4-(4-iodophenyl)butanoic acid (0.029 g, 0.1mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL) and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24a was isolated as a semi-solid(18%).

(3S,7S,21S,24S)-33-(4-iodophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25a): definted, but the a solution of compound 24a (0.025 mmol, 1eq) in dioxane (2 mL) was added 4M HCl in dioxane (2 mL). The resultingreaction mixture was stirred for 3 h at r.t. Completion of the reactionwas monitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.050 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water.

tetra-tert-butyl(2S,5S,19S,23S)-2-(4-(4-(4-bromophenyl)butanamido)butyl)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24b): To a solid mixture of 4-(4-bromophenyl)butanoic acid (0.024 g,0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL)and the mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL), and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24b was isolated as a semi-solid(6%).

(3S,7S,21S,24S)-33-(4-bromophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25b): To a solution of compound 24b (0.020 mmol, 1 eq) in dioxane(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.050 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25b.

tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-2-(4-(4-(p-tolyl)butanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24c): To a solid mixture of 4-(p-tolyl)butanoic acid (0.0178 g, 0.1mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL), and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24c was isolated as a semi-solid(18%).

(3S,7S,21S,24S)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-33-(p-tolyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25c): To a solution of compound 24c (0.03 mmol, 1 eq) in dioxane(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.050 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water.

tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-2-(4-(4-phenylbutanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24d): To a solid mixture of 4-phenylbutanoic acid (0.0164 g, 0.1 mmol)and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) and themixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1 mmol)was added to the reaction mixture and stirring was continued for 10 minat r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1 mL) wasadded dropwise at r.t and stirred at the same temperature for 12 h. DMFwas evaporated under reduced pressure to give a suspension, which wasdissolved in DCM (5 mL) and washed with water (2×10 mL), and brine (10mL). The organic layer was dried over sodium sulfate and concentrated toafford the crude product which was purified by column chromatography(SiO₂), and product 24d was isolated as a semi-solid (21%).

(3S,7S,21S,24S)-5,10,19,22,30-pentaoxo-33-phenyl-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25d): To a solution of compound 24d (0.04 mmol, 1 eq) in dioxane(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.080 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25d.

tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-2-(4-(4-phenylbutanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24e): To a solid mixture of4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid (0.023 g, 0.1mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL), and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24e was isolated as a semi-solid(17%).

(3S,7S,21S,24S)-5,10,19,22,30,33-hexaoxo-33-(5,6,7,8-tetrahydronaphthalen-2-yl)-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylicacid (25e): To a solution of compound 24e (0.02 mmol, 1 eq) in dioxane(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.080 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25e.

tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-(4-(2-(4-iodophenyl)acetamido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24f): To a solid mixture of 2-(4-Idophenyl)acetic acid (0.026 g, 0.1mmol) and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) andthe mixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1mmol) was added to the reaction mixture and stirring was continued for10 min at r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1mL) was added dropwise at r.t and stirred at the same temperature for 12h. DMF was evaporated under reduced pressure to give a suspension, whichwas dissolved in DCM (5 mL) and washed with water (2×10 mL), and brine(10 mL). The organic layer was dried over sodium sulfate andconcentrated to afford the crude product which was purified by columnchromatography (SiO₂), and product 24f was isolated as a semi-solid(10%).

(8S,11S,25S,29S)-1-(4-iodophenyl)-2,10,13,22,27-pentaoxo-11-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,9,12,21,26,28-hexaazahentriacontane-8,25,29,31-tetracarboxylicacid (25f): To a solution of compound 24f (0.02 mmol, 1 eq) in dioxane(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents was removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.080 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25f.

tetra-tert-butyl(2S,5S,19S,23S)-2-(4-(1H-indole-2-carboxamido)butyl)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,25-tetracarboxylate(24 g): To a solid mixture of indole-2-acetic acid (0.016 g, 0.1 mmol)and HATU (0.038 g, 0.1 mmol) under N₂ was added dry DMF (2 mL) and themixture was stirred for at r.t. for 5 min. DIPEA (0.017 mL, 0.1 mmol)was added to the reaction mixture and stirring was continued for 10 minat r.t. A solution of compound 23 (0.052, 0.05 mmol) in DMF (1 mL) wasadded dropwise at r.t and stirred at the same temperature for 12 h. DMFwas evaporated under reduced pressure to give a suspension, which wasdissolved in DCM (5 mL) and washed with water (2×10 mL), and brine (10mL). The organic layer was dried over sodium sulfate and concentrated toafford the crude product which was purified by column chromatography(SiO₂), and product 24 g was isolated as a semi-solid (15%).

(7S,10S,24S,28S)-1-(1H-indol-2-yl)-1,9,12,21,26-pentaoxo-10-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-2,8,11,20,25,27-hexaazatriacontane-7,24,28,30-tetracarboxylicacid (25 g): To a solution of compound 24g (0.02 mmol, 1 eq) in dioxane(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reactionmixture was stirred for 3 h at r.t. Completion of the reaction wasmonitored by TLC. Solvents were removed under reduced pressure andco-distilled with toluene (2×5 mL). The amine HCl salts were dissolvedin DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resultingreaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA(0.080 mmol, 2 eq) and distilled water (0.5 mL). Stirring was continuedfor 3 h at r.t. The reaction mixture was directly subjected to LCMSpurification using 0.1% formic acid in ACN and water to afford 25 g.

(9S,12S,26S,30S)-3,11,14,23,28-pentaoxo-1-phenyl-12-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-2-oxa-4,10,13,22,27,29-hexaazadotriacontane-9,26,30,32-tetracarboxylicacid (25h): To a solution of compound 22 (produced as defined herein)(0.02 mmol, 1 eq) in dioxane (3 mL) was added 4M HCl in dioxane (3 mL).The resulting reaction mixture was stirred for 3 h at r.t. Completion ofthe reaction was monitored by TLC. Solvents were removed under reducedpressure and co-distilled with toluene (2×5 mL). The amine HCl saltswere dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.The resulting reaction mixture was stirred for 10 min before addingp-NCS-Bn-DOTA (0.080 mmol, 2 eq) and distilled water (0.5 mL). Stirringwas continued for 3 h at r.t. The reaction mixture was directlysubjected to LCMS purification using 0.1% formic acid in ACN and waterto afford 25h.

Section 1.2 Preparation of dimethyl 4-aminopyridine-2,6-dicarboxylate(204)

Dimethyl 4-azidopyridine-2,6-dicarboxylate^([245]) (0.9445 g, 4.0 mmol),10% Pd/C (0.1419 g), and DCM:MeOH (1:1, 18 mL) were combined in around-bottom flask. After purging the flask with a balloon of H2, thereaction was stirred vigorously at room temperature under an H2atmosphere for 46 h. The gray mixture was diluted with DMF (450 mL) andfiltered through a bed of Celite. Following a subsequent filtrationthrough a 0.22 μm nylon membrane, the filtrate was concentrated at 60°C. under reduced pressure and further dried in vacuo to obtain 204 as apale-tan solid (0.824 g, 98% yield). ¹H NMR (500 MHz, DMSO-d₆): δ=7.36(s, 2H), 6.72 (s, 2H), 3.84 (s, 6H). ¹³C{¹H} APT NMR (126 MHz, DMSO-d6):δ=165.51, 156.24, 148.05, 111.99, 52.29. IR (cm⁻¹): 3409, 3339, 3230,1726, 1639, 1591, 1443, 1265, 996, 939, 787, 630, 543. HPLC t_(R)=9.369min (Method B). HRMS (m/z): 211.07213 [M+H]⁺; Calc: 211.07133.

Preparation of Ethyl 4-amino-6-(hydroxymethyl)picolinate (205)

To a refluxing suspension of 204 (0.677 g, 3.22 mmol) in absolute EtOH(27 mL) was added NaBH₄ (0.1745 g, 4.61 mmol) portionwise over 1 h togive a pale-yellow suspension. The reaction was then quenched withacetone (32 mL) and concentrated at 60° C. under reduced pressure to atan solid. The crude product was dissolved in H₂O (60 mL) and washedwith ethyl acetate (4×150 mL). The combined organics were dried oversodium sulfate and concentrated at 40° C. under reduced pressure.Further drying in vacuo yielded 205 as a pale-yellow solid (0.310 g, 49%yield).

¹H NMR (300 MHz, DMSO-d₆): δ=7.07 (d, J=2.1 Hz, 1H), 6.78 (m, 1H), 6.32(s, 2H), 5.30 (t, J=5.8 Hz, 1H), 4.39 (d, J=5.6 Hz, 2H), 4.26 (q, J=7.1Hz, 2H), 1.28 (t, J=7.1 Hz, 3H). ¹³C APT NMR (126 MHz, DMSO-d₆)δ=165.57, 162.38, 155.68, 147.25, 108.50, 107.01, 63.95, 60.61, 14.24.IR (cm⁻¹): 3439, 3217, 2974, 2917, 1717, 1643, 1600, 1465, 1396, 1378,1239, 1135, 1022, 974, 865, 783. HPLC t_(R)=8.461 min (Method B). HRMS(m/z): 197.09288 [M+H]⁺; Calc: 197.09207.

Preparation of Ethyl 4-amino-6-(chloromethyl)picolinate (206)

A mixture of thionyl chloride (2.5 mL) and 205 (0.301 g, 1.53 mmol) wasstirred in an ice bath for 1 h, and then at RT for 30 min. Theyellow-orange emulsion was concentrated at 40° C. under reduced pressureto an oily residue. The residue was neutralized with sat. aq. NaHCO₃ (12mL) and then extracted with ethyl acetate (75 mL). The organic extractwas washed with H₂O (2 mL), dried over sodium sulfate, and concentratedat 40° C. under reduced pressure. Further drying in vacuo gave 206 as anamber wax (0.287 g, 80% yield, corrected for residual ethyl acetate). ¹HNMR (500 MHz, DMSO-d₆) δ=7.18 (d, J=2.1 Hz, 1H), 6.78 (d, J=2.1 Hz, 1H),6.62 (br s, 2H), 4.62 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 1.30 (t, J=7.1Hz, 3H). ¹³C{¹H} APT NMR (126 MHz, DMSO-d₆) δ=164.75, 156.42, 156.19,147.17, 109.79, 109.50, 60.97, 46.47, 14.15. IR (cm⁻¹): 3452, 3322,3209, 2978, 2922, 1726, 1639, 1604, 1513, 1465, 1378, 1248, 1126, 1026,983, 861, 783, 752, 700. HPLC t_(R)=12.364 min (Method B). HRMS (m/z):215.05903 [M+H]⁺; Calc: 215.05818.

Preparation of Methyl6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate(209.2TFA.1H₂O)

To a clear and colorless solution of1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (7, 1.9688 g, 7.5 mmol) anddiisopropylethylamine (0.8354 g, 6.5 mmol) in dry ACN (1.075 L) at 75°C. was added dropwise a solution of 206 (0.9255 g, 5.0 mmol) in dry ACN(125 mL) over 2 h 40 min. The flask was then equipped with a condenserand drying tube, and the slightly-yellow solution was heated at refluxfor 42 h. Subsequently, the dark-gold solution containing fine, whiteprecipitate was concentrated at 60° C. under reduced pressure to anamber oil. To the crude oil was added 10% MeOH/H₂O containing 0.1% TFA(10 mL). The slight suspension was filtered, and the filtrate waspurified by preparative HPLC (Method A). Pure fractions were combined,concentrated at 60° C. under reduced pressure, and then lyophilized togive 209 (1.6350 g, 50% yield) as a pale-orange solid. ¹H NMR (500 MHz,DMSO-d₆) δ=8.75 (br s, 2H), 8.17-8.06 (m, 2H), 7.83 (dd, J=7.4, 1.5 Hz,1H), 4.68 (br s, 2H), 3.91 (s, 3H), 3.85 (br t, J=5.1 Hz, 4H), 3.69 (t,J=5.1 Hz, 4H), 3.59 (br s, 8H), 3.50 (br s, 4H), 3.23 (br t, J=5.1 Hz,4H). ¹³C{¹H} APT NMR (126 MHz, DMSO-d₆) δ 164.68, 158.78-157.98 (q,TFA), 151.44, 147.13, 139.01, 128.63, 124.87, 120.08-113.01 (q, TFA),69.33, 69.00, 65.31, 64.60, 56.43, 53.29, 52.67, 46.32. ¹⁹F NMR (470MHz, DMSO-d₆) δ=−73.84. EA Found: C, 43.88; H, 5.29; N, 6.28. Calc. forC₂₀H₃₃N₃O₆.2CF₃COOH.1H₂O: C, 43.84; H, 5.67; N, 6.39. HPLC t_(R)=12.372min (Method B). HRMS (m/z): 412.24568 [M+H]⁺; Calc: 412.24421.

Preparation of Ethyl4-amino-6-((16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate(210)

Into a round-bottom flask equipped with a condenser and drying tube wereadded 209 (0.4210 g, 0.64 mmol), Na₂CO₃ (0.3400 g, 3.2 mmol), and dryACN (10 mL). The pale-yellow suspension was heated to reflux over 15min, after which 206 (0.1508 g, 0.70 mmol, corrected for residual ethylacetate) was added as a slight suspension in dry ACN (3.5 mL). Themixture was heated at reflux for 44 h and then filtered. The orangefiltrate was concentrated at 60° C. under reduced pressure to anorange-brown oil (0.612 g), which was used in the next step withoutfurther purification. HRMS (m/z): 590.32021 [M+H]⁺; Calc: 590.31844.

Preparation of4-Amino-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinicacid (211.4TFA)

Compound 210 (0.612 g) was dissolved in 6 M HCl (7 mL) and heated at 90°C. for 17 h. The orange-brown solution containing slight precipitate wasconcentrated at 60° C. under reduced pressure to a pale-tan solid. Tothis solid was added 10% MeOH/H₂O containing 0.1% TFA (3 mL). The slightsuspension was filtered and the filtrate was purified by preparativeHPLC using Method A. Pure fractions were combined, concentrated at 60°C. under reduced pressure, and then lyophilized to give 211 as anoff-white solid (0.2974 g, 46% yield over 2 steps). ¹H NMR (500 MHz,DMSO-d₆) δ=8.13-8.08 (m, 2H), 7.80 (dd, J=7.3, 1.6 Hz, 1H), 7.64 (br s),7.24 (d, J=2.3 Hz, 1H), 6.76 (d, J=2.3 Hz, 1H), 4.74 (s, 2H), 4.15 (s,2H), 3.85 (t, J=5.0 Hz, 4H), 3.63 (t, J=5.1 Hz, 4H), 3.57-3.50 (m, 12H),3.09 (br t, J=5.2 Hz, 4H). 13C{¹H} NMR (126 MHz, DMSO-d₆) δ 165.96,163.37, 159.47, 158.78-157.98 (q, TFA), 151.93, 151.64, 148.25, 144.68,139.59, 128.43, 124.96, 120.79-113.68 (q, TFA), 109.40, 108.96, 70.03,69.89, 67.09, 65.16, 57.28, 55.85, 54.47, 53.81. ¹⁹F NMR (470 MHz,DMSO-d₆) δ=−74.03. EA Found: C, 40.60; H, 4.29; N, 7.04. Calc. forC₂₆H₃₇N₅O₄CF₃COOH: C, 40.69; H, 4.12; N, 6.98. IR (cm⁻¹): 3387, 3161,1735, 1670, 1204, 1130, 791, 722. HPLC t_(R)=11.974 min (Method B);11.546 min (Method D). HRMS (m/z): 548.26883 [M+H]+; Calc: 548.27149.

Preparation of6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinicacid (212, macropa-NCS)

A white suspension of 211 (0.1598 g, 0.16 mmol) and Na₂CO₃ (0.2540 g,2.4 mmol) was heated at reflux in acetone (10 mL) for 30 min before theslow addition of CSCl₂ (305 μL of CSCl₂, 85%, Acros Organics). Theresulting orange suspension was heated at reflux for 3 h and thenconcentrated at 30° C. under reduced pressure to a pale-orange solid.The solid was dissolved portionwise in 10% ACN/H₂O containing 0.2% TFA(8 mL total), filtered, and immediately purified by preparative HPLCusing Method C.^([246]) Pure fractions were combined, concentrated at RTunder reduced pressure to remove the organic solvent, and thenlyophilized. Fractions that were not able to be concentrated immediatelywere frozen at −80° C. Isothiocyanate 212 was obtained as a mixture ofwhite and pale-yellow solid (0.0547 g) and was stored at −80° C. in ajarof Drierite. Calculations from ¹H NMR and ¹⁹F NMR spectra of a sample of212 spiked with a known concentration of fluorobenzene estimated that212 was isolated as a tetra-TFA salt. ¹H NMR (400 MHz, DMSO-d₆)δ=8.17-8.06 (m, 2H), 8.00 (s w/fine splitting, 1H), 7.84 (d, J=1.5 Hz,1H), 7.81-7.75 (d w/fine splitting, J=7.16 Hz, 1H), 4.71 (s, 2H), 4.64(s, 2H), 3.89-3.79 (m, 8H), 3.62-3.46 (m, 16H). ¹⁹F NMR (470 MHz,DMSO-d₆) δ=−74.17. IR (cm⁻¹): ˜3500-2800, 2083, 2026, 1735, 1670, 1591,1448, 1183, 1130, 796, 717. HPLC t_(R)=15.053 min (Method B); 13.885 min(Method D). HRMS (m/z): 590.22600 [M+H]⁺; Calc: 590.22791.

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(214)

Alkyne 214 was prepared according to published methods^([247]) andisolated as an off-white powder. ¹H NMR (500 MHz, CDCl₃) δ=7.90 (s, 1H),7.58 (t, 1H, J=1.7 Hz), 7.51 (dd, 1H, J₁=8.2 Hz, J₂=1.3 Hz), 7.18 (t,1H, J=7.9 Hz), 7.05 (d, 1H, J=7.7 Hz), 6.38 (d, 1H, J=7.9 Hz), 6.28 (brs, 1H), 5.77 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 4.02 (m, 1H), 3.53 (m,1H), 3.05 (m, 1H), 3.00 (s, 1H), 2.39 (m, 2H), 2.07 (m, 1H), 1.88 (m,1H), 1.74 (m, 1H), 1.62 (m, 1H), 1.49-1.37 (m, 4H), 1.41 (s, 18H), 1.37(s, 9H).

Preparation of 2,5-Dioxopyrrolidin-1-ylN²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysinate(215)

A suspension of Fmoc-L-Lys(Boc)-OH (5.0 g, 10.7 mmol) andN,N-disuccinimidyl carbonate (2.74 g, 10.7 mmol) in CH₂Cl₂ (50 mL) wasstirred at room temperature under argon. Then DIPEA (1.86 mL, 10.7 mmol)was added, and the suspension was stirred overnight. The solvent wasevaporated under reduced pressure and the crude product was purified byflash chromatography (0-100% EtOAc in hexane). Lysine 215 was isolatedas a white powder (2.5 g, 41%). ¹H NMR (500 MHz, CDCl₃) δ=7.76 (d, 2H,J=7.6 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t, 2H, J=7.4 Hz), 7.32 (t, 2H,J=7.3 Hz), 5.46 (br s, 1H), 4.71 (m, 2H), 4.45 (m, 2H), 4.23 (t, 1H,J=6.6 Hz), 3.14 (br s, 2H), 2.85 (s, 4H), 2.02 (m, 1H), 1.92 (m, 1H),1.58 (m, 4H), 1.44 (s, 9H).

Preparation of tert-ButylN²—(N²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(216)

A suspension of L-Lys(Z)-OtBu HCl (1.49 g, 4.0 mmol) in CH₂Cl₂ (15 mL)was treated with DIPEA (0.87 mL, 5.0 mmol). To the resulting mixture wasadded a solution of lysine 215 (2.2 g, 3.9 mmol) in CH₂Cl₂ (10 mL), andthe reaction was stirred overnight at room temperature under argon. Itwas then washed with saturated NaCl solution, and the organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure. Thecrude product was purified by flash chromatography (0-100% EtOAc inhexane), and di-lysine 216 was isolated as a white powder (2.2 g, 72%).¹H NMR (500 MHz, CDCl₃) δ=7.76 (d, 2H, J=7.5 Hz), 7.59 (d, 2H, J=7.3Hz), 7.40 (t, 2H, J=7.5 Hz), 7.32 (m, 8H), 6.69 (br s, 1H), 5.60 (br s,1H), 5.06 (m, 4H), 4.72 (br s, 1H), 4.43 (m, 1H), 4.38 (m, 1H), 4.21 (m,1H), 3.14 (m, 4H), 1.85 (m, 2H), 1.73 (m, 2H), 1.50 (m, 4H), 1.46 (s,9H), 1.44 (s, 9H), 1.39 (m, 4H).

Preparation of 2,5-Dioxopyrrolidin-1-yl 2-(4-iodophenyl)acetate (217)

A solution of 2-(4-iodophenyl)acetic acid (786 mg, 3.0 mmol) and EDC HCl(671 mg, 3.5 mmol) in CH₂Cl₂ (20 mL) was stirred for 15 min at roomtemperature under argon. Then N-hydroxysuccinimide (368 mg, 3.2 mmol)and NEt₃ (0.56 mL, 4.0 mmol) were added and the reaction was stirred for7 h. It was then washed with saturated NaCl solution, and the organiclayer was dried over MgSO₄, filtered and concentrated under reducedpressure. The crude residue was purified by flash chromatography (0-100%EtOAc in hexane), and the NHS ester 217 was isolated as a white solid(760 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ=7.69 (d, 2H, J=7.9 Hz), 7.09(d, 2H, J=7.9 Hz), 3.88 (s, 2H), 2.83 (s, 4H).

Preparation of tert-ButylN²—(N²-(1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(218)

To a solution of Fmoc-protected di-lysine 216 (768 mg, 0.97 mmol) inCH₂Cl₂ (4 mL) was added NHEt₂ (2.07 mL, 20 mmol). The solution wasstirred overnight at room temperature. The solvents were removed underreduced pressure, and the crude product, a yellow oil, was used withoutfurther purification. To a solution of this oil (183 mg, 0.32 mmol) inCH₂Cl₂ (3 mL) were added successively solutions of NEt₃ (57 μL, 0.41mmol) in CH₂Cl₂ (1 mL) and azido-PEG₆-NHS ester (100 mg, 0.21 mmol;Broadpharm, USA) in CH₂Cl₂ (1 mL), and the reaction was stirredovernight at room temperature. It was then diluted with CH₂Cl₂ andwashed successively with H₂O and saturated NaCl solution. The organiclayer was dried over MgSO₄, filtered and concentrated under reducedpressure to give azide 218 as a colorless oil (184 mg; 95%) without needfor further purification. Mass (ESI+): 926.4 [M+H]⁺. Calc. Mass=925.54.

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2,17,20,23,26,29,32-heptaoxa-4,10,13-triazatetratriacontan-34-yl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(219)

A solution of 100 μL of 0.5 M CuSO₄ and 100 μL of 1.5 M sodium ascorbatein DMF (0.5 mL) was mixed for 5 min and was then added to a solution of218 (184 mg, 0.20 mmol) and 214 (132 mg, 0.21 mmol) in DMF (2.5 mL). Theresulting mixture was stirred at room temperature for 45 min. It wasthen concentrated under reduced pressure and the crude residue waspurified by flash chromatography (0-30% MeOH in EtOAc) to give triazole219 as an orange oil (285 mg; 87%). Mass (ESI+): 1557.2 [M+H]⁺. Calc.Mass=1555.90.

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((23S,26S)-26-(tert-butoxycarbonyl)-23-(4-((tert-butoxycarbonyl)amino)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(220)

Cbz-Protected triazole 219 (285 mg, 0.18 mmol) was dissolved in MeOH (15mL) in a two-neck flask. To the solution was added 10% Pd/C (20 mg), andthe suspension was shaken and the flask evacuated. The suspension wasthen placed under an H₂ atmosphere and stirred overnight. It wasfiltered through celite, and the filter cake was washed three times withMeOH. The combined filtrate was concentrated under reduced pressure togive the free amine as a colorless oil (117 mg; 45%) that was usedwithout further purification. Mass (ESI+): 1423.8 [M+H]⁺. Calc.Mass=1422.77. To a solution of the amine (117 mg, 82 μmol) in CH₂Cl₂ (4mL) was added a solution of DIPEA (23 μL, 131 mmol) in CH₂Cl₂ (1 mL),and the mixture was stirred at room temperature under argon. Then asolution of 217 (37 mg, 103 μmol) in CH₂Cl₂ (2 mL) was added, and thereaction was stirred at room temperature for 2 h. It was then pouredinto H₂O (10 mL) and the layers were separated. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure togive the crude product as a colorless semi-solid. The crude product waspurified by prep TLC (10% MeOH in EtOAc) to give phenyl iodide 220 as acolorless oil (34 mg; 25%). Mass (ESI+): 1666.6 [M+H]⁺. Calc.Mass=1665.80.

Preparation of(((S)-1-Carboxy-5-(3-(3-(1-((23S,26S)-26-carboxy-23-(4-(3-(2-carboxy-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)pyridin-4-yl)thioureido)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (221, macropa-RPS-070)

To a solution of 220 (34 mg, 20 μmol) in CH₂Cl₂ (2 mL) was added TFA(0.5 mL), and the reaction was stirred at room temperature for 5 h. Itwas then concentrated under reduced pressure, and the crude product wasdiluted with H₂O and lyophilized to give the free amine as a TFA salt.Mass (ESI+): 1342.5 [M+H]⁺. Mass (ESI−): 1340.6 [M−H]⁻. Calc.Mass=1341.50. To a solution of the amine (9 mg, 6.7 μmol) in DMF (0.5mL) was added a solution of macropa-NCS 212 (15 mg, 25.4 μmol) in DMF(0.5 mL). Then DIPEA (300 μL, 1.72 mmol) was added and the reaction wasstirred at room temperature for 2 h. The volatiles were removed underreduced pressure and the crude product was purified by prep HPLC to givemacropa-RPS-070 (221) as a white powder (5.4 mg; 42%). Mass (ESI+):1932.76 [M+H]⁺. 1931.09 [M+H]⁻. Calc. Mass=1931.91.

Preparation of Radiosynthesis of ²²⁵Ac-macropa-RPS-070

General. All reagents were purchased from Sigma Aldrich unless otherwisenoted, and were reagent grade. Hydrochloric acid (HCl) was traceSELECT®(>99.999%) for trace analysis quality. Aluminum-backed silica thin layerchromatography (TLC) plates were purchased from Sigma Aldrich. Stocksolutions of 0.05 M HCl and 1 M NH₄₀Ac were prepared by dilution inMilli-Q® water.

Radiolabeling Procedure. To a solution of ²²⁵Ac(NO₃)₃ (Oak RidgeNational Laboratory, USA) in 0.05 M HCl (17.9 MBq in 970 μL) was added20 μL of a 1 mg/mL solution of macropa-RPS-070 in DMSO. The pH wasraised to 5-5.5 by addition of 90 μL M NH₄₀Ac. The reaction was allowedto stand at room temperature for 20 min with periodic shaking. Then, 200μL of the reaction solution was removed and diluted with 3.8 mL ofnormal saline (0.9% NaCl in deionized H₂O; VWR) to give a solution witha concentration of 910 kBq/mL. An aliquot was removed from the finalsolution and spotted onto an aluminum-backed silica TLC plate todetermine radiochemical yield. An aliquot of the ²²⁵Ac(NO₃)₃ solution in0.05M HCl was spotted in a parallel lane as a control. The plate wasimmediately run in a 10% v/v MeOH/10 mM EDTA mobile phase, and thenallowed to stand for 8 h to enable radiochemical equilibrium to bereached. The plate was visualized on a Cyclone Plus Storage PhosphorSystem (Perkin Elmer) following a 3-min exposure on the phosphor screen.The radiochemical yield was expressed as a ratio of²²⁵Ac-macropa-RPS-070 to total activity and was determined to be 98.1%.

Biodistribution Studies with ²²⁵Ac-macropa-RPS-070.

Cell Culture. The PSMA-expressing human prostate cancer cell line,LNCaP, was obtained from the American Type Culture Collection. Cellculture supplies were from Invitrogen unless otherwise noted. LNCaPcells were maintained in RPMI-1640 medium supplemented with 10% fetalbovine serum (Hyclone), 4 mM L-glutamine, 1 mM sodium pyruvate, 10 mMN-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5 mg/mLD-glucose, and 50 μg/mL gentamicin in a humidified incubator at 37°C./5% CO₂. Cells were removed from flasks for passage or for transfer to12-well assay plates by incubating them with 0.25%trypsin/ethylenediaminetetraacetic acid (EDTA).

Inoculation of Mice with Xenografts. All animal studies were approved bythe Institutional Animal Care and Use Committee of Weill CornellMedicine and were undertaken in accordance with the guidelines set forthby the USPHS Policy on Humane Care and Use of Laboratory Animals.Animals were housed under standard conditions in approved facilitieswith 12 h light/dark cycles. Food and water was provided ad libitumthroughout the course of the studies. Hairless male nu/nu mice werepurchased from the Jackson Laboratory. For inoculation in mice, LNCaPcells were suspended at 4×10⁷ cells/mL in a 1:1 mixture of PBS:Matrigel(BD Biosciences). Each mouse was injected in the left flank with 0.25 mLof the cell suspension. Biodistributions were conducted when tumors werein the range 100-400 mm³.

Biodistribution of ²²Ac-macropa-RPS-070 in LNCaP xenograft mice. FifteenLNCaP xenograft tumor-bearing mice (5 per time point) were injectedintravenously with a bolus injection of 85-95 kBq and 100 ng (50 μmol)of each ligand. The mice were sacrificed by cervical dislocation at 4,24 and 96 h post injection. A blood sample was removed, and a fullbiodistribution study was conducted on the following organs (withcontents): heart, lungs, liver, small intestine, large intestine,stomach, spleen, pancreas, kidneys, muscle, bone, and tumor. Tissueswere weighed and counted on a 2470 Wizard Automatic Gamma Counter(Perkin Elmer). 1% ID/mL samples were counted prior to and followingeach set of tissue samples to enable decay correction to be undertaken.Counts were corrected for decay and for activity injected, and tissueuptake was expressed as percent injected dose per gram (% ID/g).Standard error measurement was calculated for each data point.

TABLE 1 Organ distribution of ²²⁵Ac-macropa-RPS-070 at t = 4 h, 24 h,and 96 h following intravenous injection in LNCaP xenograft mice (n = 5per time point). Values expressed as % ID/g. 1 2 3 4 5 Mean SEM 4 hBlood 0.90654 0.55246 1.11808 0.8276 0.65638 0.81221 0.0986 Heart0.75759 0.65317 0.77395 0.75148 0.6565 0.71894 0.02604 Lungs 0.995580.60669 1.2979 0.98587 0.88664 0.94691 0.10516 Liver 1.62187 1.346321.74207 1.68077 1.3957 1.55735 0.0788 Small Intestine 0.1998 0.162820.3104 0.24413 0.17094 0.21762 0.02721 Large Intestine 1.36298 0.651621.27419 0.91656 0.81901 1.00487 0.13563 Stomach 0.33963 0.2471 0.304170.4109 0.21221 0.3028 0.03489 Spleen 1.40902 0.70804 1.61264 1.108150.8756 1.14269 0.16632 Pancreas 0.55487 0.41637 0.55317 0.4675 0.66040.53047 0.04182 Kidneys 65.5884 20.5274 108.233 33.654 33.0707 52.214615.8618 Muscle 0.68006 0.80579 0.72817 0.67666 0.65617 0.70937 0.026884Bone 1.14861 1.12335 1.48731 0.92036 1.15463 1.16685 0.09106 Tumor6.73177 10.7309 23.8367 15.3682 7.50352 12.8342 3.1429 24 h Blood0.34825 0.31324 0.22083 0.29453 0.27697 0.29076 0.0211 Heart 0.522560.56334 0.4521 0.47914 0.46483 0.49639 0.02052 Lungs 0.53778 0.450770.46083 0.4286 0.44831 0.46526 0.01887 Liver 1.57844 1.47552 1.137761.14264 1.48473 1.36382 0.09305 Small Intestine 0.08784 0.09914 0.088220.09466 0.10375 0.09473 0.00309 Large Intestine 0.13296 0.1259 0.132520.13425 0.13176 0.13148 0.00145 Stomach 0.1296 0.12119 0.1119 0.146750.15329 0.13255 0.00773 Spleen 0.62075 0.65764 0.62013 0.57685 0.585540.61218 0.01443 Pancreas 0.39847 0.39119 0.50347 0.33315 0.31944 0.389140.03252 Kidneys 4.98792 4.25707 3.94586 3.66457 4.10348 4.19178 0.22185Muscle 0.61193 0.5149 0.44832 0.78028 0.44579 0.56025 0.06276 Bone1.27255 1.06645 0.83943 1.00576 0.69755 0.97635 0.09828 Tumor 11.61639.26927 7.50158 4.41446 8.04683 8.16969 1.17583 96 h Blood 0.190420.19188 0.15206 0.16528 0.23822 0.18757 0.01475 Heart 0.39939 0.423980.42861 0.445863 0.45595 0.43331 0.01098 Lungs 0.30165 0.50912 0.469440.37811 0.36979 0.40562 0.03717 Liver 0.79406 0.8144 0.73301 0.79170.79415 0.78546 0.01374 Small Intestine 0.04372 0.0577 0.03752 0.044310.04136 0.04492 0.00341 Large Intestine 0.04349 0.09663 0.04522 0.041980.03927 0.05332 0.01087 Stomach 0.03442 0.04708 0.03448 0.02845 0.023660.03362 0.00393 Spleen 0.48373 0.394 0.44261 0.43481 0.53966 0.458960.02469 Pancreas 0.09848 0.37696 0.30549 0.31625 0.33352 0.28614 0.04847Kidneys 1.30286 1.3239 2.00405 1.39866 1.45955 1.4978 0.12958 Muscle0.3022 0.52492 0.25089 0.29815 0.2528 0.32579 0.05095 Bone 0.863910.86874 0.83831 1.12223 0.82042 0.90272 0.05557 Tumor 4.04259 4.077996.73954 4.58107 4.84503 4.85724 0.49449

Discussion of Results for Above-Described of [²²⁵Ac(macropa)]⁺ Complexes

The in vivo stability of [²²⁵Ac(macropa)]⁺ was assessed by comparing itsbiodistribution to those of ²²⁵Ac(NO₃)₃ and [²²⁵Ac(DOTA)]⁻. C57BL/6 micewere injected via tail vein with 10-50 kBq of each radiometal complexand were sacrificed after 15 min, 1 h, or 5 h. The amount of ²²⁵Acretained in each organ was quantified by gamma counting and reported asthe percent of injected dose per gram of tissue (% ID/g). Inadequatestability of an ²²⁵Ac complex leading to the loss of radioisotope invivo is manifested by the accumulation of ²²⁵Ac in the liver, spleen,and bone of mice.^([11,12,31]) The biodistribution profile ofuncomplexed ²²⁵Ac(NO₃)₃ (FIG. 5A) reveals slow blood clearance andexcretion, coupled to large accumulation in the liver and spleen. Thebiodistribution profile of [²²⁵Ac(macropa)]⁺ (FIG. 5B) differs markedlyfrom that of ²²⁵Ac(NO₃)₃.

[²²⁵Ac(macropa)]⁺ was rapidly cleared from mice, with very littleactivity measured in blood by 1 h post injection. Most of the injecteddose was renally excreted and subsequently detected in the urine, whichexplains the moderate kidney and bladder uptake of [²²⁵Ac(macropa)]⁺observed in mice at 15 min and 1 h post injection. Of significance,[²²⁵Ac(macropa)]⁺ did not accumulate in any organ over the time courseof the study, indicating that the complex does not release free ²²⁵Ac³⁺in vivo. Its biodistribution profile was similar to that of[²²⁵Ac(DOTA)]⁻ (FIG. 5C), which has been previously shown to retain²²⁵Ac³⁺ in-vivo.^([7]) Notably, [²²⁵Ac(DOTA)]⁻ appeared to clear morerapidly through the urine and was taken up to a lesser extent in thethyroid. These differences may arise in part due to the opposite chargesof the complexes. Collectively, the results of these biodistributionstudies demonstrate that [²²⁵Ac(macropa)]⁺ is highly stable in vivo.

RPS-070 was conjugated to macropa-NCS, where this construct bears aglutamate-urea-lysine moiety that inhibits the prostate-specificmembrane antigen (PSMA), [²³⁷⁻²⁴¹] a membrane-bound glycoprotein that isoverexpressed in prostate cancer cells.[²⁴²] An albumin-bindingfunctional group, in this case the group including iodophenyl, is also acritical component of these compounds that prolongs their circulationhalf-life.[^(243,244)] Radiolabeling of macropa-RPS-070 with ²²⁵Acproceeded in 20 min at RT and pH 5-5.5 to give a RCY of 98%.²²⁵Ac-macropa-RPS-070 (85-95 kBq) was then injected into LNCaP (prostatecancer) tumor xenograft-bearing mice, and the biodistribution of thecomplex was determined at 4, 24, and 96 h post injection (Table 1 supra,FIG. 6). ²²⁵Ac-macropa-RPS-070 was rapidly cleared from the blood andprimarily distributed to the kidneys and tumor (52±16% ID/g and 13±3%ID/g, respectively, at 4 h post injection). After 4 h, most of theactivity cleared from the kidneys and gradual tumor washout wasobserved. Importantly, the complex exhibited negligible uptake by otherorgans (<1% ID/g at 96 h post injection) and did not amass in any organover time. The activity that cleared from the tumor from 4-96 h remainedchelated by macropa-RPS-070, as evidenced by the lack of accumulation of²²⁵Ac in the liver, spleen, and bone of mice during this time. Theseresults are significant because they demonstrate that macropa-RPS-070can stably retain ²²⁵Ac in vivo over several days and that the constructcan be selectively targeted to tumors.

Section 1.2 References

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Section 1.3

General Methods. All solvents were purchased from Sigma Aldrich and wereof reagent grade quality unless otherwise indicated. Solvents were driedeither by distillation over an activated stainless steel column (PureProcess Technology, LLC) column or by drying over activated molecularsieves. Reagents were purchased from Sigma Aldrich, except for2-azidoacetic acid-NHS ester and the azido-PEGn-NHS ester compounds,which were purchased from BroadPharm. The reagents were all of reagentgrade and were used without any further purification.

All reactions described below were carried out in dried glassware.Purifications were performed using silica chromatography on VWR® HighPurity Silica Gel 60 Å, preparative TLC on silica-coated glass plates(Analtech) and by flash chromatography using a CombiFlash Rf+(TeledyneIsco) system. Preparative HPLC was performed using an XBridge™ Prep C185 μm OBD™ 19×100 mm column (Waters) on a dual pump Agilent ProStar HPLCfitted with an Agilent ProStar 325 Dual Wavelength UV-Vis Detector. UVabsorption was monitored at 220 nm and 280 nm. A binary solvent systemwas used, with solvent A comprising H₂O+0.01% TFA and solvent Bconsisting of 90% v/v MeCN/H₂O+0.01% TFA. Purification was achievedusing the following gradient HPLC method: 0% B 0-1 min., 0-100% B 1-28mins., 100-0% B 28-30 mins.

Final products were identified and characterized using thin layerchromatography, analytical HPLC and mass spectrometry. NMR spectroscopywas used to confirm the structure of compounds 7a, 8a, 26 and 28.Analytical HPLC was performed using an XSelect™ CSH™ C18 5 m 4.6×50 mmcolumn (Waters). Mass determinations were performed by LCMS analysisusing a Waters ACQUITY UPLC® coupled to a Waters SQ Detector 2. NMRanalyses were performed using a Bruker Avance III 500 MHz spectrometer.Spectra are reported as ppm and are referenced to the solvent resonancesin chloroform-d (Sigma Aldrich). The purity of all compounds evaluatedin the biological assay was >95% as judged by analytical HPLC.

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(26)

Alkyne 26 was prepared according to the protocols described in Kelly J,Amor-Coarasa A, Nikolopoulou A, Kim D, Williams C., Jr, Ponnala S,Babich J W. Synthesis and pre-clinical evaluation of a new class ofhigh-affinity ¹⁸F-labeled PSMA ligands for detection of prostate cancerby PET imaging. Eur J Nucl Med Mol Imaging 2017; 44:647-61 and isolatedas an off-white powder. ¹H NMR (500 MHz, CDCl₃) δ 7.90 (s, 1H), 7.58 (t,1H, J=1.7 Hz), 7.51 (dd, 1H, J₁=8.2 Hz, J₂=1.3 Hz), 7.18 (t, 1H, J=7.9Hz), 7.05 (d, 1H, J=7.7 Hz), 6.38 (d, 1H, J=7.9 Hz), 6.28 (br s, 1H),5.77 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 4.02 (m, 1H), 3.53 (m, 1H), 3.05(m, 1H), 3.00 (s, 1H), 2.39 (m, 2H), 2.07 (m, 1H), 1.88 (m, 1H), 1.74(m, 1H), 1.62 (m, 1H), 1.49-1.37 (m, 4H), 1.41 (s, 18H), 1.37 (s, 9H).

Synthetic Procedure to that in Section 1.1 for tert-butylN²—(N²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(8a)

2,5-dioxopyrrolidin-1-ylN²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysinate(7a): A suspension of Fmoc-L-Lys(Boc)-OH 6a (5.0 g, 10.7 mmol) andN,N′-disuccinimidyl carbonate (2.74 g, 10.7 mmol) in CH₂Cl₂ (50 mL) wasstirred at room temperature under argon. Then DIPEA (1.86 mL, 10.7 mmol)was added, and the suspension was stirred overnight. The solvent wasevaporated under reduced pressure and the crude product was purified byflash chromatography (0-100% EtOAc in hexane). The NHS ester 7a wasisolated as a white powder (2.5 g, 41%). ¹H NMR (500 MHz, CDCl₃) δ 7.76(d, 2H, J=7.6 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t, 2H, J=7.4 Hz), 7.32(t, 2H, J=7.3 Hz), 5.46 (br s, 1H), 4.71 (m, 2H), 4.45 (m, 2H), 4.23 (t,1H, J=6.6 Hz), 3.14 (br s, 2H), 2.85 (s, 4H), 2.02 (m, 1H), 1.92 (m,1H), 1.58 (m, 4H), 1.44 (s, 9H).

tert-butylN²—(N²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(8a): A suspension of L-Lys(Z)-OtBu.HCl (1.49 g, 4.0 mmol) in CH₂Cl₂ (15mL) was treated with DIPEA (0.87 mL, 5.0 mmol). To the resulting mixturewas added a solution of compound 7a (2.2 g, 3.9 mmol) in CH₂Cl₂ (10 mL),and the reaction was stirred overnight at room temperature under argon.It was then washed with saturated NaCl solution, and the organic layerwas dried over MgSO₄, filtered and concentrated under reduced pressure.The crude product was purified by flash chromatography (0-100% EtOAc inhexane), and di-lysine 8a was isolated as a white powder (2.2 g, 72%).¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, 2H, J=7.5 Hz), 7.59 (d, 2H, J=7.3Hz), 7.40 (t, 2H, J=7.5 Hz), 7.32 (m, 8H), 6.69 (br s, 1H), 5.60 (br s,1H), 5.06 (m, 4H), 4.72 (br s, 1H), 4.43 (m, 1H), 4.38 (m, 1H), 4.21 (m,1H), 3.14 (m, 4H), 1.85 (m, 2H), 1.73 (m, 2H), 1.50 (m, 4H), 1.46 (s,9H), 1.44 (s, 9H), 1.39 (m, 4H).

2,5-dioxopyrrolidin-1-yl 2-(4-iodophenyl)acetate (28)

A solution of 2-(4-iodophenyl)acetic acid 27 (786 mg, 3.0 mmol) andEDC.HCl (671 mg, 3.5 mmol) in CH₂Cl₂ (20 mL) was stirred for 15 min atroom temperature under argon. Then N-hydroxysuccinimide (368 mg, 3.2mmol) and TEA (0.56 mL, 4.0 mmol) were added and the reaction wasstirred for 7 h. It was then washed with saturated NaCl solution, andthe organic layer was dried over MgSO₄, filtered and concentrated underreduced pressure. The crude residue was purified by flash chromatography(0-100% EtOAc in hexane), and the NHS ester 28 was isolated as a whitesolid (760 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ 7.69 (d, 2H, J=7.9 Hz),7.09 (d, 2H, J=7.9 Hz), 3.88 (s, 2H), 2.83 (s, 4H).

Synthesis of trifunctional ligands (RPS-061, RPS-063, RPS-066, RPS-067,RPS-068, RPS-069) with representative procedure for synthesis of RPS-069

tert-butylN²—(N²-(1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(29e): To a solution of Fmoc-protected compound 8a (768 mg, 0.97 mmol)in CH₂Cl₂ (4 mL) was added diethylamine (2.07 mL, 20 mmol). The solutionwas stirred overnight at room temperature. The solvents were removedunder reduced pressure, and the crude product, a yellow oil, was usedwithout further purification. To a solution of this yellow oil (183 mg,0.32 mmol) in CH₂Cl₂ (3 mL) were added solutions of TEA (57 μL, 0.41mmol) in CH₂Cl₂ (1 mL) and azido-PEG₆-NHS ester (100 mg, 0.21 mmol) inCH₂Cl₂ (1 mL), and the reaction was stirred overnight at roomtemperature. It was then diluted with CH₂Cl₂ and washed successivelywith H₂O and saturated NaCl solution. The organic layer was dried overMgSO₄, filtered and concentrated under reduced pressure to give azide29e as a colorless oil (184 mg; 95%) without need for furtherpurification. Mass (ESI+): 926.4 [M+H]⁺. Calc. Mass=925.54.

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2,17,20,23,26,29,32-heptaoxa-4,10,13-triazatetratriacontan-34-yl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(30e): A solution of 100 μL 0.5M CuSO₄ and 100 μL 1.5 M sodium ascorbatein DMF (0.5 mL) was mixed for 5 min and was then added to a solution of29e (184 mg, 0.20 mmol) and 26 (132 mg, 0.21 mmol) in DMF (2.5 mL). Theresulting mixture was stirred at room temperature for 45 min. It wasthen concentrated under reduced pressure and the crude residue waspurified by flash chromatography (0-30% MeOH in EtOAc) to give triazole30e as an orange oil (285 mg; 87%). Mass (ESI+): 1557.2 [M+H]⁺. Calc.Mass=1555.90.

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((23S,26S)-26-(tert-butoxycarbonyl)-23-(4-((tert-butoxycarbonyl)amino)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(31e): Cbz-Protected triazole 30e (285 mg, 0.18 mmol) was dissolved inMeOH (15 mL) in a two-neck flask. To the solution was added 10% Pd/C (20mg), and the suspension was shaken and the flask evacuated. Thesuspension was then placed under H2 atmosphere and stirred overnight. Itwas filtered through celite, and the filter cake was washed three timeswith MeOH. The combined filtrate was concentrated under reduced pressureto give the free amine as a colorless oil (117 mg; 45%) that was usedwithout further purification. Mass (ESI+): 1423.8 [M+H]⁺. Calc.Mass=1422.77. To a solution of the free amine (117 mg, 82 μmol) inCH₂Cl₂ (4 mL) was added a solution of DIPEA (23 μL, 131 mmol) in CH₂Cl₂(1 mL), and the mixture was stirred at room temperature under argon.Then a solution of 28 (37 mg, 103 μmol) in CH₂Cl₂ (2 mL) was added, andthe reaction was stirred at room temperature for 2 h. It was then pouredinto H₂O (10 mL) and the layers were separated. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure togive the crude product as a colorless semi-solid. The crude product waspurified by prep TLC (10% MeOH in EtOAc) to give phenyl iodide 31e as acolorless oil (34 mg; 25%). Mass (ESI+): 1666.6 [M+H]⁺. Calc.Mass=1665.80.

(((1S)-1-carboxy-5-(3-(3-(1-((23S,26S)-26-carboxy-33-(4-iodophenyl)-21,24,32-trioxo-23-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-069): To a solution of 31e (34 mg, 20 μmol) in CH₂Cl₂ (2 mL)was added TFA (0.5 mL), and the reaction was stirred at room temperaturefor 5 h. It was then concentrated under reduced pressure and the crudeproduct was diluted in H₂O and lyophilized to give the free amine as aTFA salt. Mass (ESI+): 1342.5 [M+H]⁺. Mass (ESI−): 1340.6 [M−H]⁻. Calc.Mass=1341.50. To a solution of p-SCN-Bn-DOTA-2.5HCl.2.5H₂O(Macrocyclics, Inc.) (13 mg, 19 μmol) in H₂O (0.5 mL) was added asolution of the free amine (18 mg, 13 μmol) in DMF (1 mL). DIPEA wasadded until the reaction was pH 9). The reaction was stirred at roomtemperature for 3 h, at which point the reaction mixture was thenpurified by prep HPLC. The peak corresponding to the desired product wascollected and lyophilized to give RPS-069 as a white powder (8 mg; 32%).Mass (ESI+): 1893.3 [M+H]⁺, 947.6 [(M+2H)/2]⁺. Mass (ESI−): 1891.4[M−H]⁻, 945.5 [(M−2H)/2]⁻. Calc. Mass=1892.70.

(((1S)-1-carboxy-5-(3-(3-(1-((17S,20S)-20-carboxy-27-(4-iodophenyl)-15,18,26-trioxo-17-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12-tetraoxa-16,19,25-triazaheptacosyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-061): RPS-061 was synthesized from the common building blocks26, 8a and 28 and azido-PEG₄-NHS ester according to the proceduredescribed for RPS-069. Mass (ESI+): 1805.6664 [M+H]⁺. Calc.Mass=1804.6594.

(((1S)-1-carboxy-5-(3-(3-(1-((14S,17S)-17-carboxy-24-(4-iodophenyl)-12,15,23-trioxo-14-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9-trioxa-13,16,22-triazatetracosyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-063): RPS-063 was synthesized from the common building blocks26, 8a and 28 and azido-PEG₃-NHS ester according to the proceduredescribed for RPS-069. Mass (ESI+): 1762.4 [M+H]⁺. Mass (ESI−): 1760.5[M−H]⁻. Calc. Mass=1761.71.

(((1S)-1-carboxy-5-(3-(3-(1-((29S,32S)-32-carboxy-39-(4-iodophenyl)-27,30,38-trioxo-29-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18,21,24-octaoxa-28,31,37-triazanonatriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-066): RPS-066 was synthesized from the common building blocks26, 8a and 28 and azido-PEG₈-NHS ester according to the proceduredescribed for RPS-069. Mass (ESI+): 1982.3 [M+H]⁺, 991.5 [(M+2H)/2]⁻.Calc. Mass=1980.76.

(((1S)-1-carboxy-5-(3-(3-(1-((41S,44S)-44-carboxy-51-(4-iodophenyl)-39,42,50-trioxo-41-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40,43,49-triazahenpentacontyl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-067): RPS-067 was synthesized from the common building blocks26, 8a and 28 and azido-PEG₁₂-NHS ester according to the proceduredescribed for RPS-069. Mass (ESI+): 1079.7 [(M+2H)/2]⁺. Mass (ESI−):2155.6 (M−H)⁻, 1077.7 [(M−2H)/2]-. Calc. Mass=2156.86.

Synthesis of RPS-068

tert-butylN²—(N²-(2-azidoacetyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(32): To a solution of Fmoc-protected 8a (768 mg, 0.97 mmol) in CH₂Cl₂(4 mL) was added diethylamine (2.07 mL, 20 mmol). The solution wasstirred overnight at room temperature. The solvents were removed underreduced pressure, and the crude product, the free amine as a yellow oil,was used without further purification. To a solution of free amine (356mg, 0.63 mmol) in CH₂Cl₂ (6 mL) was added a solution of TEA (175 μL,1.26 mmol) in CH₂Cl₂ (1 mL), and the resulting mixture was stirred atroom temperature. Then a solution of 2-azidoacetic acid NHS ester (138mg, 0.69 mmol) in CH₂Cl₂ (3 mL) was added, and the reaction was stirredat room temperature. After 3 h, it was diluted with CH₂Cl₂ and washedsuccessively with H₂O and saturated NaCl solution. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure togive pale yellow azide 32 (374 mg, 92%) that was used without furtherpurification. Mass (ESI+): 648.1 [M+H]⁺. Calc. Mass=647.36.

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2-oxa-4,10,13-triazapentadecan-15-yl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(33): A solution of 150 μL 0.5M CuSO₄ and 150 μL 1.5M sodium ascorbatein DMF (0.2 mL) was mixed for 5 min and was then added to a solution ofazide 32 (374 mg, 0.54 mmol) and alkyne 26 (358 mg, 0.54 mmol) in DMF (2mL). The mixture was stirred at room temperature for 2 h before thesolvent was removed under reduced pressure. The resulting residue wasdissolved in CH₂Cl₂ and washed with H₂O. The organic layer was driedover MgSO₄, filtered and concentrated under reduced pressure. The crudeproduct was purified by flash chromatography (0-10% MeOH in EtOAc), buta small impurity remained. Therefore a second purification was performedby prep TLC (100% EtOAc), and the product was isolated as a colorlessoil (146 mg; 21%). Mass (ESI+): 1278.6 [M+H]⁺. Calc. Mass=1277.73.

di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-(2-(((10S,13S)-13-(tert-butoxycarbonyl)-20-(4-iodophenyl)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-triazaicosan-10-yl)amino)-2-oxoethyl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(34): Triazole 33 (146 mg, 0.11 mmol) was dissolved in MeOH (10 mL) in atwo-neck flask. To the solution was added 10% Pd/C (10 mg), and thesuspension was shaken while the flask was evacuated. Then the suspensionwas stirred under H2 atmosphere for 2 h before the mixture was filteredthrough celite. The filter cake was washed three times with MeOH and thefiltrates were combined and concentrated under reduced pressure to givethe free amine as a black residue (91 mg; 72%) that contained traces ofminor impurities. The crude product was used without furtherpurification. Mass (ESI+): 1144.6 [M+H]⁺. Calc. Mass=1143.69. To asolution of free amine (90 mg, 79 μmol) and NEt₃ (14 μL, 150 μmol) inCH₂Cl₂ (4 mL) was added a solution of 28 (36 mg, 100 μmol) in CH₂Cl₂ (1mL). The resulting mixture was stirred overnight at room temperature,then it was diluted with CH₂Cl₂ and washed successively with H₂O andsaturated NaCl solution. The organic layer was dried over MgSO₄,filtered and concentrated under reduced pressure to give a blackresidue. The residue was dissolved in EtOAc, and a black precipitate wasremoved by filtration. The resulting crude product was purified by prepTLC (5% MeOH in EtOAc), and phenyl iodide 34 was isolated as a whitesolid (21 mg; 19%). Mass (ESI+): 1388.4 [M+H]⁺. Calc. Mass=1387.63.

(((1S)-1-carboxy-5-(3-(3-(1-(2-(((2S)-1-(((S)-1-carboxy-5-(2-(4-iodophenyl)acetamido)pentyl)amino)-1-oxo-6-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)hexan-2-yl)amino)-2-oxoethyl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (RPS-068): To a solution of 34 (20 mg, 15 mol) in CH₂Cl₂ (3.5 mL)was added TFA (0.5 mL). The reaction was stirred at room temperature for4 h, then it was concentrated under reduced pressure. The crude residuewas dissolved in H₂O and lyophilized to give the free amine as a TFAsalt. Mass (ESI+): 1064.1 [M+H]⁺. Calc. Mass=1063.33. To a solution ofp-SCN-Bn-DOTA-2.5HCl.2.5H₂O (Macrocyclics, Inc.) (10 mg, 15 μmol) in 1mL 50% DMF in H₂O was added a solution of free amine (16 mg, 15 μmol) inDMF (0.7 mL). NEt₃ was added (110 μL) until the pH of the reaction wasapproximately 9. The reaction was stirred for 1 h, then the reactionmixture was purified by prep HPLC. The peak corresponding to the productwas collected and lyophilized to give RPS-068 as a white powder (2.4 mg;10%). Mass (ESI+): 1615.2 [M+H]⁺. Mass (ESI−): 1613.3 [M−H]⁻, 806.4[(M−2H)/2]⁻. Calc. Mass=1614.53.

Radiochemistry

General Methods: All reagents were purchased from Sigma Aldrich unlessotherwise noted, and were reagent grade. Hydrochloric acid (HCl) andsodium acetate (NaOAc) were of traceSELECT® (>99.999%) quality. Allwater (H2O) used was highly pure (18 mΩ). Analytical IPLC was performedon a dual-pump Varian Dynamax IPLC (Agilent Technologies) fitted with adual UV-Vis detector, and radiochemical purity was determined using aNaI(Tl) flow count detector (Bioscan). UV absorption was monitored at220 nm and 280 nm. Solvent A was 0.01% trifluoroacetic acid (TFA) in H₂Oand solvent B was 0.01% TFA in 90% v/v acetonitrile (MeCN):H₂O. Analyseswere performed on a Symmetry C18 4.6×50 mm, 100-A column (Waters) at aflow rate of 2 mL/min and a gradient of 0% B to 100% B over 10 minutes.

Production of Ga-66: Gallium-66 (t_(1/2)=9.4 h) was produced from theirradiation of a natural zinc target (Alfa Aesar; 0.5 g, 100 μmthickness, 99.999%) by a (p,n) reaction over 2 h using a 15 MeV beam anda 17.5 mA current. The irradiation of natural zinc produces Ga-66, Ga-67and Ga-68. The target was left overnight to allow Ga-68 (t_(1/2)=68 min)to decay before processing. The principal radionuclidic impurity duringprocessing was Ga-67 (t_(1/2)=78.3 h), at approximately 3%. The targetwas dissolved in conc. HCl (5 mL) and the ⁶⁶Ga³⁺ ions were separatedfrom Zn²⁺ ions by 20 mg UTEVA anion exchange (Eichrom) according topreviously published methods [20]. The column was later washed twicewith 3 ml of a 5M HCl solution to eliminate the excess Zn²⁺. Finally,the purified ⁶⁶Ga³⁺ ions were eluted with H₂O (0.5 mL), leading to afinal solution containing 2.14-2.36 GBq/mL (58-64 mCi/mL) andapproximately 0.1 M HCl.

Radiolabeling of RPS series: ⁶⁶Ga-Labeled ligands were preparedaccording to the following procedure. 100 μL of the Ga-66 stock solutioncontaining 167-205 MBq (4.5-5.5 mCi) was diluted with 1 mL 0.05M HCl. Tothis solution was added 40-80 μL of a 1 mg/mL solution of precursor inDMSO. The reaction was initiated by addition of 40 μL 3N NaOAc, and thesolution was mixed at 95° C. on an Eppendorf ThermoMixer® C (VWR) for 25min. The mixture was then diluted with H₂O and passed through apre-activated Sep-Pak C18 Plus Light cartridge (Waters). The cartridgewas washed with H₂O and the product was eluted with 100 μL EtOH (300proof, VWR) followed by 900 μL saline (0.9% NaCl solution; VWR). Finalradioactivity concentrations were in the range 7.4-85 MBq/mL (0.2-2.3mCi/mL), and radiochemical purity was greater than 90%.

Labeling with Lu-177: No-carrier-added Lu-177 (EndolucinBeta®) waspurchased from iTG (Garching, Germany) as the chloride salt, with anactivity at calibration of 1.5-3.0 GBq (40-80 mCi). An aliquotcontaining 0.52-0.93 GBq (14-25 mCi) of the Lu-177 stock solution wasdiluted to 1 mL with 0.05M HCl. To this solution was added 20 μg ofprecursor as a 1 mg/mL solution in DMSO. The reaction was initiated byraising the pH to 4-5 using 3N NaOAc (20-30 μL). The buffered solutionwas heated for 10 min at 95° C. on an analog heating block (VWR). Afterthe solution had cooled to room temperature, it was diluted with H₂O (9mL) and passed through a pre-activated Sep-Pak C18 Plus Light cartridge(Waters). The cartridge was washed with H₂O (5 mL) and the product waseluted with 500 μL EtOH (200 proof, VWR) followed by 500 μL saline (0.9%NaCl solution; VWR). An aliquot (40-98 μL) was removed from thissolution and diluted to 4 mL with saline. The final concentration ofeach ligand in the injected solution was 0.23-0.28 μM, with a range ofactivity of 3.5-8.8 MBq/mL (93-240 μCi/mL). The specific activity of the¹⁷⁷Lu-labeled compounds ranged from 15.8-48.8 GBq/μmol. Radiochemicalyields were 33-80% after purification and reformulation, andradiochemical purity was greater than 98%.

Cell Culture: The PSMA expressing human prostate cancer cell line,LNCaP, was obtained from the American Type Culture Collection. Cellculture supplies were obtained from Invitrogen unless otherwise noted.LNCaP cells were maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum (Hyclone), 4 mM L-glutamine, 1 mM sodium pyruvate, 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5 mg/mLD-glucose, and 50 μg/mL gentamicin in a humidified incubator at 37°C./5% CO₂. Cells were removed from flasks for passage or for transfer to12-well assay plates by incubating them with 0.25%trypsin/ethylenediaminetetraacetic acid (EDTA).

In vitro determination of IC₅₀: IC₅₀ values of the non-labeled,metal-free ligands were determined by screening in a multi-concentrationcompetitive binding assay against^(99m)Tc-((7S,12S,16S)-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-2-((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)-9,14-dioxo-2,8,13,15-tetraazaoctadecane-7,12,16,18-tetracarboxylicacid technetium tricarbonyl complex) (^(99m)Tc-MIP-1427) for binding toPSMA on LNCaP cells, according to previously described methods [18,19]with small modifications. Briefly, LNCaP cells were plated 48 h prior tothe experiment to achieve a density of approximately 5×10⁵ cells/well(in triplicate) in RPMI-1640 medium supplemented with 0.25% bovine serumalbumin. The cells were incubated for 2 h with 1 nM ^(99m)Tc-MIP-1427 inserum-free RPMI-1640 medium in the presence of 0.001-10,000 nM testcompounds. Radioactive incubation media was then removed by pipette andthe cells were washed twice using 1 mL ice-cold PBS 1× solution. Cellswere harvested from the plates following treatment with 1 mL 1M NaOH andtransferred to tubes for radioactive counting using a 2470 Wizard²Automatic Gamma Counter (Perkin Elmer). Standard solutions (10% ofactivity added to each well) were prepared to enable decay correction.IC₅₀ values were determined by fitting the data points to a sigmoidalHills1 curve in Origin software.

Inoculation of mice with xenografts: All animal studies were approved bythe Institutional Animal Care and Use Committee of Weill CornellMedicine and were undertaken in accordance with the guidelines set forthby the USPHS Policy on Humane Care and Use of Laboratory Animals.Animals were housed under standard conditions in approved facilitieswith 12 h light/dark cycles. Food and water was provided ad libitumthroughout the course of the studies. Hairless male nu/nu mice werepurchased from the Jackson Laboratory. For inoculation in mice, LNCaPcells were suspended at 4×10⁷ cells/mL in a 1:1 mixture of PBS:Matrigel(BD Biosciences). Each mouse was injected in the left flank with 0.25 mLof the cell suspension. The mice were imaged when the tumors reachedapproximately 200-400 mm³, while biodistributions were conducted whentumors were in the range 100-400 mm³.

Imaging of ⁶⁶Ga-RPS ligands in LNCaP xenograft mice: LNCaP xenografttumor-bearing mice (2-3 per compound) injected intravenously with abolus injection of 0.56-5.4 MBq (15-145 μCi) of the ⁶⁶Ga-labeled ligand.The specific activity of the tracers was in the range 14.8-47 MBq/μmol(0.4-1.27 mCi/μmol). The mice were imaged using PET/CT (Inveon™; SiemensMedical Solutions, Inc.) at 1, 3, 6 and 24 h post-injection followinginhalation anesthetization with isoflurane. Total acquisition time was30 min for the 1 h, 3 h and 6 h images, and 60 min for 24 h time point.A CT scan was obtained immediately before the acquisition for bothanatomical co-registration and attenuation correction. Images werereconstructed using the Inveon™ software supplied by the vendor.Image-derived tumor and kidney uptake was estimated by comparison to a10% injected dose per cubic mm (% ID/mm³) standard introduced into theimaging field of view. The standard was prepared by dilution of 10% ofthe injected activity to 1 mL with saline. Volumes of interest (VOIs)were drawn with the aid of the CT and confirmed by PET. The contents ofthe VOIs were integrated and the calculated counts were converted to %ID/mm³ by direct comparison to the aforementioned standard followingcorrection for activity injected.

Biodistribution studies of 177Lu-labeled ligands in LNCaP xenograftmice: LNCaP xenograft tumor-bearing mice (5 per time point per compound)were injected intravenously with a bolus injection of 348-851 kBq(9.4-23 μCi) and 37-50 ng (23-25 μmol) of each ligand. The mice weresacrificed at 4, 24 and 96 h post injection. A blood sample was removed,and a full biodistribution study was conducted on the following organs(with contents): heart, lungs, liver, small intestine, large intestine,stomach, spleen, pancreas, kidneys, muscle, bone and tumor. Tissues wereweighed and counted on a 2470 Wizard² Automatic Gamma Counter (PerkinElmer). Counts were corrected for decay and for activity injected, andtissue uptake was expressed as percent injected dose per gram (% ID/g).Standard error measurement was calculated for each data point.

Dosimetry: The dosimetry was calculated assuming a linear interpolationbetween the three time points. The average injected dose per organ wascalculated by using the average of the activity and organ weights of themice at that time point. Intermediate time points at every 4 hours weregenerated using the linear approximation and all time points werecorrected for decay during the time interval between points. Thesecurves were integrated using a trapezoidal approximation and the sumused to determine the residence time.

Statistical Analysis: A comprehensive statistical analysis was performedto compare the tissue uptake of each compound across time. The normalityassumption was visually checked by a quantile-quantile (QQ) plot, and alog transformation was applied to the data to remove the skew effect.Under each organ and each compound, a one-way ANOVA (Analysis ofVariance) with Tukey's honestly significant difference (HSD) post-hoctest was used to evaluate the difference in measurement across threetime points. An overall P-value under an F-test and pairwise ones undert-test was determined. Furthermore, a two-way ANOVA was used to assessthe influence of time, compound and their interaction in each organ. TheP-values are reported. A confidence interval of 95% was used todetermine statistical significance.

Section 1.3 Results and Discussion

The three moieties were linked by an azide-derivatizedpolyethyleneglycol (PEG) spacer incorporating 0 (RPS-068), 3 (RPS-063),4 (RPS-061), 6 (RPS-069), 8 (RPS-066) or 12 (RPS-067) PEG subunits (seeTable 2). No degradation or decomposition of the ligands was observedover the course of three months during storage at 4° C. as determined byanalytical HPLC. In contrast, similar analogues in which a Gly-Gly-Glylinker or a C₇H₁₄ linker was used in place of the PEG spacer were foundto decompose over the course of a few weeks under the same storageconditions.

In an effort to minimize the use of animals, an initial screening of thecompounds was performed using μPET/CT imaging to avoid unnecessarytesting. For this purpose, Ga-66 was selected in preference to other PETradionuclides such as Ga-68 or Sc-44 due to its longer half-life(t_(1/2)=9.4 h) and the possibility of producing larger quantities(>1.85 GBq/50 mCi) in the cyclotron. Greater than 99% of the Ga-66 wasrecovered in the purification process, but labeling yields remainedconsistently low (46.4±20.5%, n=7). The variable and low labeling yieldswere likely due to the presence of Zn²⁺ ions in the labeling reactiondue to incomplete separation of the ⁶⁶Ga³⁺ ions from the dissolvedtarget material.

The ¹⁷⁷Lu-labeled constructs were prepared in 67±17% (n=20)radiochemical yield following purification and reformulation. Variationin final product yield was largely due to differences in trappingefficiency by the C18 cartridge, with ¹⁷⁷Lu-RPS-067 and ¹⁷⁷Lu-RPS-068showing the lowest trapping (approximately 40%). Labeling yields priorto purification were typically >75% for all ligands as determined byradioHPLC. There was no apparent correlation between PEG length andlabeling yield. The Lu-177 labeled ligands were stable to radiolysis for24 h when stored at 4° C. Radiochemical stability was not determined atroom temperature.

The total amount of ligand injected was 22-24 μmol per mouse in order toremain proportional to clinical mass doses of ¹⁷⁷Lu-PSMA-617administered to human subjects [4]. The specific activity of thepreparations ranged from 15.8-48.8 GBq/μmol, consistent with the valuesreported for the preclinical evaluation of ¹⁷⁷Lu-PSMA-617 [21]. The massof ⁶⁶Ga-labeled ligands injected was 4 μg per mouse, corresponding to1.8-2.5 nmol. This greater mass was required to account for the poorerlabeling yields with this radionuclide.

All compounds were evaluated for PSMA binding in vitro using acell-based competitive binding assay. All compounds were highly potent(IC₅₀<10 nM), validating our selection of the 3-ethynylphenylureaderivative of Glu-urea-Lys as the PSMA-targeting pharmacophore. Therange of affinities was defined by RPS-063 (IC₅₀=1.5 0.3 nM) and RPS-067(IC₅₀=9.5 1.1 nM) (Table 2). Potency generally decreased with increasingPEG linker length, although RPS-068 (PEG₀; IC₅₀=2.1±0.1 nM) was slightlyless potent than RPS-063. In the same assay the IC₅₀ of PSMA-617 wasdetermined to be 6.6±0.7 nM (Table 2), consistent with the previouslyreported value [21].

TABLE 2 Summary of compound structures and key in vitro and in vivocharacteristics. IC₅₀ values were determined by a competitive bindingassay in LNCaP cells. Tumor uptake was determined by biodistributionstudies with the corresponding ¹⁷⁷Lu-labeled compound in LNCaP xenografttumor-bearing mice. (a = 4 h p.i.; b = 24 h p.i.) Max. Mol. Tumor Wt.IC₅₀ Uptake^(a) Cpd. PEG Structure (g/mol) (nM) (% ID/g) PSMA- 617 n.a.

1042.15 6.6 ± 0.7 14.4 ± 2.5 RPS- 068 0

1615.52 2.1 ± 0.1 26.9 ± 2.0^(b)

RPS- 3 n = 2  1761.71 1.5 ± 0.3 30.0 ± 6.9 063 RPS- 4 n = 3  1805.76 4.1± 0.9 20.4 ± 3.1 061 RPS- 6 n = 5  1893.87 3.8 ± 0.4 17.0 ± 2.1 069 RPS-8 n = 7  1981.97 5.2 ± 0.4 18.7 ± 1.1 066 RPS- 12  n = 11 2158.18 9.5 ±1.1  7.6 ± 1.2 067

Preliminary screening of the compounds in mice was performed by PEVCTimaging using ⁶⁶Ga (FIG. 7) with the intention of avoiding fullbiodistribution studies on compounds that showed poor targeting. Imageswere analyzed to determine quantitative uptake in the tumor and kidneysat 1, 3, 6 and 24 h post injection. In the tumor, uptake was high butdecreased with increasing PEG length. ⁶⁶Ga-RPS-068 (PEG0; maximum uptakeof 9.7±2.0% ID/cm³ (3 h); 8.3±2.8% ID/cm³ at 24 h) and ⁶⁶Ga-RPS-063(PEG3; maximum uptake of 9.5±2.4% ID/cm³ (6 h); 7.9±3.0% ID/cm³ at 24 h)showed the greatest uptake, with ⁶⁶Ga-RPS-061 (PEG4; 6.1±1.1% ID/cm³)and ⁶⁶Ga-RPS-069 (PEG6; 7.0±3.9% ID/cm³) demonstrating comparable uptakeat 24 h post injection. ⁶⁶Ga-RPS-066 (PEG8; maximum uptake of 7.8±0.7%ID/cm³ (3 h); 5.5±0.4% ID/cm³ at 24 h) and ⁶⁶Ga-RPS-067 (PEG12; maximumuptake of 6.6±3.2% ID/cm³ (1 h); 3.1±1.7% ID/cm³ at 24 h) showed loweruptake at all time points, but still exceeded ⁶⁶Ga-PSMA-617 (maximumuptake of 3.1±0.4% ID/cm³ (1 h); 1.1±0.4% ID/cm³ at 24 h. Kidney uptakewas generally on the same order as tumor uptake and was greatest at 1 hpost injection. Uptake ranged from 10.5±2.1% ID/cm³ (⁶⁶Ga-RPS-061) to3.7±0.5% ID/cm³ (⁶⁶Ga-RPS-067) at 1 h post injection, and from 1.9±0.3%ID/cm³ (⁶⁶Ga-RPS-068) to 0.2±0.1% ID/cm³ (⁶⁶Ga-RPS-066 and ⁶⁶Ga-RPS-067)at 24 h post injection. In comparison, the maximum kidney uptake of⁶⁶Ga-PSMA-617 was 0.4±0.1% ID/cm³, while uptake at 24 h was 0.1±0.1%ID/cm³.

Following the promising imaging studies, biodistribution studies of the¹⁷⁷Lu-labeled ligands confirmed the trends evident in the PET images.Although the affinity of the compounds for PSMA is clustered within oneorder of magnitude, the tissue distribution of the ligands showedconsiderable variation. This was most evident in the tissues that areknown to express PSMA, including the tumor and the kidney (FIG. 8).Tumor uptake was high and remained high for ¹⁷⁷Lu-RPS-068 (PEG₀),¹⁷⁷Lu-RPS-063 (PEG3), ¹⁷⁷Lu-RPS-061 (PEG4), ¹⁷⁷Lu-RPS-069 (PEG6) and¹⁷⁷Lu-RPS-066 (PEG₈). For the higher affinity compounds ¹⁷⁷Lu-RPS-068and ¹⁷⁷Lu-RPS-063, uptake at 4 h p.i. was 21.8±2.8% ID/g and 30.0±3.1%ID/g, respectively, with 14.9±1.5% ID/g and 12.9±0.5% ID/g stillremaining at 96 h p.i. Clearance was not statistically significant by 24h (p>0.13). Uptake of ¹⁷⁷Lu-RPS-069 and ¹⁷⁷Lu-RPS-066 was 17.0±2.1% ID/gand 18.7±1.1% ID/g respectively at 4 h p.i. and decreased to 9.8±0.8%ID/g and 5.9±0.7% ID/g at 96 h p.i. Nevertheless, these uptake valuesare significantly greater after 24 h p.i. than those observed for¹⁷⁷Lu-PSMA-617 (14.4±1.1% ID/g and 3.5±0.3% ID/g at 4 h and 96 h p.i.,p<0.001). ¹⁷⁷Lu-RPS-067 (PEG12), the lowest affinity ligand, accumulatedat only 7.6±1.2% ID/g at 4 h p.i. and had cleared to 3.2±0.1% ID/g at 96h p.i. This uptake was significantly lower than all other ligands(p<0.001) except ¹⁷⁷Lu-PSMA-617.

A similar trend within the RPS series was observed for kidney uptake,with the lowest affinity ligand, ¹⁷⁷Lu-RPS-067, distinguished bysignificantly lower uptake (54.9±13.2% ID/g) at 4 h p.i. than the otherRPS ligands tested (p<0.004) (FIG. 8). Kidney uptake exceeded 100% ID/gat 4 h p.i. for all other ligands of the RPS series, while¹⁷⁷Lu-PSMA-617 was found to clear rapidly (14.1±3.1% ID/g at 4 h p.i.)in agreement with published reports [21]. Prolonged retention of¹⁷⁷Lu-RPS-068 (87.3±6.7% ID/g at 24 h p.i.) and ¹⁷⁷Lu-RPS-063 (51.8±8.6%ID/g at 24 h p.i.) was evident, but ¹⁷⁷Lu-RPS-066 (6.2±0.8% ID/g at 24 hp.i.) and ¹⁷⁷Lu-RPS-067 (4.6±0.6% ID/g at 24 h p.i.) clearedsignificantly (p<0.001) and more rapidly. Uptake of these two ligandswas significantly lower than the other RPS ligands (p<0.001) but notsignificantly different to each other (p<0.14).

In combination with persistent tumor accumulation, more rapid kidneyclearance gave rise to tumor-to-kidney ratios of 1.92±0.30 and 1.25±0.20for ¹⁷⁷Lu-RPS-066 and ¹⁷⁷Lu-RPS-067 at 24 h p.i. These ratios aresignificantly higher than the other RPS ligands (p<0.001), but reflectlow and rapid kidney clearance rather than high and persistent tumoruptake. For the same reason, the tumor-to-kidney ratio of ¹⁷⁷Lu-PSMA-617is significantly higher than the other ligands at all time pointsstudied (p<0.001). By 96 h, each member of the RPS series demonstrated atumor-to-kidney ratio substantially in excess of 1 (range=1.56-3.32).

Uptake in other tissues was negligible with the exception of the spleen,which showed modest, likely PSMA-mediated uptake at 4 h p.i. followed byclearance to background levels (FIG. 8). As expected, blood activity wassignificantly greater for all of the RPS series than for ¹⁷⁷Lu-PSMA-617(p<0.05). ¹⁷⁷Lu-RPS-063, ¹⁷⁷Lu-RPS-061 and ¹⁷⁷Lu-RPS-068 showed thehighest blood activity at 4 h p.i. (FIG. 9), while ¹⁷⁷Lu-RPS-069,¹⁷⁷Lu-RPS-066 and ¹⁷⁷Lu-RPS-067 showed lower blood retention at the sametime point. By 24 h p.i., the blood activity was below 0.3% ID/g for allof the ligands, and by 96 h it had decreased to below 0.1% ID/g.Interestingly, although all of the RPS ligands contained the samealbumin-binding group, N⁶-(2-(4-iodophenyl)acetyl)-L-lysine, significantdifferences (p<0.001) were observed between the shorter PEG compounds¹⁷⁷Lu-RPS-068 and ¹⁷⁷Lu-RPS-063 and the longer PEG compounds¹⁷⁷Lu-RPS-066 and ¹⁷⁷Lu-RPS-067. This indicates that the linkerinfluences binding to plasma proteins and/or clearance.

The inverse correlation between PEG length and affinity for PSMA isconsistent with findings reported to date for PSMA constructs and othertargeting ligands. Small PEG linkers such as PEG3 or PEG4 have beenincorporated into small molecule drug conjugates that target PSMA[22,23], but constructs of this nature to date have shown low affinityand/or poor tumor uptake. One SAR study did establish that PEG2 and PEG4linkers best retained PSMA affinity in a family of PSMA-targetingcontrast agents, with PEG12 and PEG24 leading to large decreases inaffinity [24]. These results were in agreement with the observation thata PEG12 linker decreased affinity relative to PEG8 in a small moleculeGCPII ligand [25]. An SAR study of the influence of PEG linkers on⁶⁸Ga-labeled antagonists of bombesin found that affinity slightlyweakened upon each incremental extension of the PEG linker [26]. Thisstudy also identified small differences in the biodistribution of theligands.

The areas under the curve (AUC) of the time-activity curves (TACs) fortumor uptake suggest that the ¹⁷⁷Lu-labeled RPS-061, -063, 066, -068 and-069ligands deliver a significantly larger dose to the tumor than does¹⁷⁷Lu-PSMA-617 (FIG. 10). This is confirmed by a comparison of the doseintegrals in the tumor. ¹⁷⁷Lu-RPS-068 and ¹⁷⁷Lu-RPS-063 are nearly fourtimes higher than ¹⁷⁷Lu-PSMA-617, while ¹⁷⁷Lu-RPS-061, ¹⁷⁷Lu-RPS-069 and¹⁷⁷Lu-RPS-066 are also at least two times higher (FIG. 11).

It is likely that the tumor uptake of the ¹⁷⁷Lu-labeled RPS ligands isalso higher than other ¹⁷⁷Lu-labeled PSMA-targeting ligands reported todate, including ¹⁷⁷Lu-PSMA I&T, with a reported uptake in LNCaP tumorsof 7.96±1.76 at 1 h p.i. [27], and the recently reported ¹⁷⁷Lu-CTT1403,which was reported to reach 46% ID/g at 72 h p.i. in PC3-PIP tumors[23]. PSMA expression is PC3-PIP tumors is higher than typically foundin human prostate cancers, notably ten-fold greater than LNCaP cells[28], meaning that uptake of ¹⁷⁷Lu-CTT1403 is likely to be considerablylower in LNCaP tumors. Uptake in LNCaP tumors for ¹⁷⁷Lu-RPS-063 and¹⁷⁷Lu-RPS-068 is on a par with the uptake reported for ¹³¹1-MIP-1095[29], the small molecule, to our knowledge, with the greatest uptake inLNCaP xenograft tumors reported to date. A comparison of the TACs for¹⁷⁷Lu-RPS-063, ¹⁷⁷Lu-RPS-068 and ¹³¹1-MIP-1095 suggests a similar AUCfor the 96 h period studied (FIG. 10).

It has previously been reported that prolonged blood retention leads toincreased tumor accumulation with time [23,30], a consequence presumablyof an increase in the number of times the ligand passes through thetumor bed. ¹⁷⁷Lu-RPS-068 appears to increase from 4 h to 24 h, but thisdifference is not statistically significant (p=0.26). The phenomenon isnot evident among the other trifunctional RPS ligand series after 4 hp.i. either, though it is possible that delayed blood clearance duringthe first 4 h may increase tumor uptake in this time interval.Nevertheless, clearance of the ligands from the tumor is slow, enablingthe delivery of greater amounts of activity to the target tissuecompared to ¹⁷⁷Lu-PSMA-617. Further modification of albumin binding bysubstitution of the albumin binding group may be used to subtly modifyblood clearance and reinforce the high and persistent tumoraccumulation.

In spite of promising clinical outcomes using PSMA-targeted radioligandtherapy for mCRPC, next generation ligands that (1) overcome resistanceto P-particle radiation, (2) are appropriate for treating diffusemetastatic lesions (particularly in the bone) and (3) provide longerduration of progression free survival are essential to continuedimprovements in treatment. Although minimal toxicity is currentlyassociated with a single administration of ¹⁷⁷Lu-PSMA-617 or¹³¹I-MIP-1095, the incidence of hematological toxicity and persistentxerostomia can increase upon subsequent therapy cycles [3] whilebiochemical response may decrease [3,5]. Alpha-particle mediated therapyhas been proposed as a method of overcoming resistance to P-particlesand reducing hematological toxicity [31]. Early preclinical studies with²¹³Bi-PSMA I&T have identified the formation of DNA double-strand breaksin tumors in vivo [32], while preliminary treatment of human patientswith ²²⁵Ac-PSMA-617 or ²¹³Bi-PSMA-617 have led to dramatic responses inrefractory cancer [31,33]. Nevertheless, multiple therapy cycles wererequired for efficacy, leading to irreversible xerostomia andkeratoconjunctivitis sicca [33].

These early findings have demonstrated the therapeutic potential forα-particle radiotherapy, but highlighted the need for radioligands withgreater therapeutic index that can deliver a high dose to the tumor.Each of ¹⁷⁷Lu-RPS-061, -063, -066, -068 and -069 shows significantlyhigher tumor uptake than ¹⁷⁷Lu-PSMA-617 in LNCaP xenograft tumors, withthe corresponding increase in AUC correlating to an increase in the doseof radioactivity delivered to the tumor. The tumor-to-kidney ratios of¹⁷⁷Lu-RPS-063, ¹⁷⁷Lu-RPS-068 and ¹⁷⁷Lu-RPS-061, the three ligands withhighest tumor uptake, are 2.75±0.17, 1.56±0.23 and 3.64±0.29,respectively, at 96 h p.i. In contrast, ¹⁷⁷Lu-CTT1403 never reaches 1.0[23]. Although the tumor-to-kidney ratio of ¹⁷⁷Lu-PSMA-617 at the sametime point is 14.39±2.2, and the ratio of tumor dose integral to kidneydose integral over the 96 h is 1.95, these are driven by very low kidneyuptake rather than high tumor uptake. It has been widely demonstratedthat the expression of PSMA in the kidneys of nude mice is higher thanexpression levels in human kidneys [34,35,36], meaning that preclinicalstudies consistently overestimate the dose delivered to this organ.Several PSMA-targeted therapeutics including ¹³¹I-MIP-1095 (29),¹⁷⁷Lu-DKFZ-617 (21) and ¹⁷⁷Lu-PSMA I&T (37) all show early kidneyconcentrations at or above 100% ID/g in nude mice, yet have been safelytranslated to the clinic with acceptable; albeit not identical kidneydoses.

Furthermore, additional nephroprotection schemes, includingpharmacological displacement with 2-PMPA, have been shown to reduceactivity in the kidney still further [37,38]. Taken together, theseobservations illustrate that the trifunctional RPS ligands show both thehigh and persistent tumor uptake and broad therapeutic index that aredesirable for α-particle radiotherapeutics.

Section 1.3 References

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Section 1.4

Materials and Instrumentation. The synthesis of RPS-074 is describedbelow. All solvents and reagents were purchased from commercial vendorsand used without further purification. The intermediate di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(406) and macropa-NCS were synthesized as described above. Compoundswere purified using silica chromatography on VWR® High Purity Silica Gel60 Å, preparative TLC on silica-coated glass plates (Analtech), or flashchromatography using a CombiFlash Rf+(Teledyne Isco) system. PreparativeHPLC was performed using an XBridge™ Prep C18 5 μm OBD™ 19×100 mm column(Waters) on a dual pump Agilent ProStar HPLC fitted with an AgilentProStar 325 Dual Wavelength UV-Vis Detector. UV absorption was monitoredat 220 nm and 280 nm. A binary solvent system was used, with solvent Acomprising H₂O+0.01% TFA and solvent B consisting of 90% v/vMeCN/H₂O+0.01% TFA. Purification was achieved using the followinggradient HPLC method: 0% B 0-1 min., 0-100% B 1-28 mins., 100-0% B 28-30mins.

Final products were identified and characterized using thin layerchromatography, analytical HPLC and mass spectrometry. NMR spectroscopywas used to confirm the structure of compound 406 and macropa-NCS. NMRanalyses were performed using a Bruker Avance III 500 MHz spectrometer.Spectra are reported in CDCl₃ or DMSO-d6. Analytical HPLC was performedusing an XSelect™ CSH™ C18 5 μm 4.6×50 mm column (Waters). Massdeterminations were performed by LCMS analysis using a Waters ACQUITYUPLC® coupled to a Waters SQ Detector 2. The purity of all compoundsevaluated in the biological assay was >95% purity as judged byanalytical HPLC.

Synthesis of macropa-RPS-074

Preparation of tert-ButylN²-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(402). To a stirred mixture of Fmoc-N-amido-PEG-8-acid (663 mg, 1.0mmol), L-N^(ε)-Z-Lys-OtBu hydrochloride (446 mg, 1.2 mmol) and HATU (456mg, 1.2 mmol) in DMF (10 mL) was added DIPEA (260 mg, 2.0 mmol), and thereaction was stirred overnight at room temperature under N₂. The solventwas removed under reduced pressure and the crude residue was purified byflash chromatography (0-10% MeOH in CH₂Cl₂) to give compound 2 as acolorless oil (845 mg, 86%). Mass (ESI+): 983.0 [M+H]⁺. Calc. Mass:981.5.

Preparation of tert-ButylN²-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oyl)-N⁶-(4-(4-iodophenyl)butanoyl)-L-lysinate(403). Compound 402 (1.45 g, 1.48 mmol) was dissolved in MeOH (25 mL).10% Palladium on charcoal (15 mg) was added, and the suspension wasstirred in a three-neck flask at room temperature for 10 min. The flaskwas evacuated and then placed under an H2 atmosphere. The suspension wasthen stirred at room temperature for 5 h before it was filtered throughcelite. The filter cake was washed with MeOH, and the combined filtratewas concentrated under reduced pressure to give the amine as a yellowoil (1.17 g, 93%) that was used without further purification. Mass(ESI+): 849.4 [M+H]⁺. Calc. Mass: 848.0. To a solution of the amine (865mg, 1.01 mmol) and 2,5-dioxopyrrolidin-1-yl 4-(4-iodophenyl)butanoate(387 mg, 1.00 mmol) in CH₂Cl₂ (20 mL) was added TEA (167 μL, 1.20 mmol).The resulting solution was stirred at room temperature under Ar for 4 h.The solution was then washed successively with 1% v/v AcOH/H₂O andbrine. The organic layer was dried over MgSO₄, filtered and concentratedunder reduced pressure to give a yellow oil. The crude product waspurified by flash chromatography (0-30% MeOH in CH₂Cl₂) and compound 3was isolated as a yellow oil (360 mg, 32%). Mass (ESI+): 1120.9 [M+H]⁺.Calc. Mass: 1119.5.

Preparation of tert-ButylN²—((S)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazanonatriacontan-39-oyl)-N⁶-(4-(4-iodophenyl)butanoyl)-L-lysinate(404). A solution of 403 (360 mg, 0.32 mmol) and diethylamine (0.67 mL,6.48 mmol) in CH₂Cl₂ (2 mL) was stirred at room temperature for 7 h. Thesolution was concentrated under reduced pressure and the crude residuewas purified by flash chromatography (0-30% MeOH in CH₂Cl₂). Thefractions containing the product were combined and concentrated to givethe amine as a yellow oil (96 mg, 33%). Mass (ESI+): 899.2 [M+H]⁺. Calc.Mass: 897.4. To a solution of the amine (96 mg, 107 μmol) andFmoc-L-Lys(Boc)-OSu (62 mg, 110 μmol) in CH₂Cl₂ (5 mL) was added TEA (28μL, 200 μmol). The mixture was stirred overnight at room temperatureunder Ar. The solvent was removed under reduced pressure and the crudeproduct was purified by flash chromatography (0-30% MeOH in CH₂Cl₂). Thedesired product co-eluted with a minor impurity, therefore the mixturewas purified a second time by prep TLC (10% v/v MeOH/CH₂Cl₂). Compound404 was isolated as a colorless oil (78 mg, 51%). Mass (ESI+): 1349.0[M+H]⁺. Calc. Mass: 1347.6.

Preparation of tert-ButyN²—((S)-10-amino-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazanonatriacontan-39-oyl)-N-(4-(4-iodophenyl)butanoyl)-L-lysinate(405). A solution of 404 (73 mg, 54 μmol) and diethylamine (0.5 mL, 4.83mmol) in CH₂Cl₂ (2 mL) was stirred overnight at room temperature. Thesolvent was removed under reduced pressure and the crude product wasdissolved in MeOH and purified by prep TLC (10% v/v MeOH in CH₂Cl₂).Amine 405 was isolated as a pale oil (25 mg, 41%). Mass (ESI+): 1127.7[M+H]⁺. Calc. Mass: 1126.2.

Preparation of di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-(2-(2-(2-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(407). A solution of 0.5M CuSO₄ (100 μL) and 1.5M sodium ascorbate (100μL) was mixed until the brown color was converted to orange. Thismixture was then added to a solution of 406 (315 mg, 0.5 mmol) andazido-PEG3-NHS (177 mg, 0.5 mmol) in DMF (2 mL). The mixture was stirredat room temperature for 2 h. It was then diluted with CH₂Cl₂ and washedwith H₂O. The organic layer was dried over MgSO₄, filtered andconcentrated under reduced pressure to give a pale oil. The crudeproduct was purified by flash chromatography (0-30% MeOH in CH₂Cl₂) togive compound 407 as a clear oil (460 mg, 95%). Mass (ESI+): 975.9[M+H]⁺. Calc. Mass: 974.5.

Preparation of di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((14S,45S)-45-(tert-butoxycarbonyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-54-(4-iodophenyl)-12,15,43,51-tetraoxo-3,6,9,19,22,25,28,31,34,37,40-undecaoxa-13,16,44,50-tetraazatetrapentacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(408). To a solution of amine 405 (25 mg, 22 μmol) in CH₂Cl₂ (4 mL) wasadded a solution of ester 407 (24 mg, 25 μmol) and TEA (7 μL, 50 μmol)in CH₂Cl₂ (1 mL). The reaction was stirred for 5 h at room temperatureunder Ar. Then the reaction was concentrated under reduced pressure andthe crude residue was dissolved in EtOAc (1 mL) and purified by prep TLC(90% EtOAc in hexanes) to give compound 408 as a pale oil (33 mg, 76%).Mass (ESI+): 994.3 [(M+2H)/2]⁺. Calc. Mass: 1986.2.

Preparation of(((S)-1-Carboxy-5-(3-(3-(1-((14S,45S)-45-carboxy-14-(4-(3-(2-carboxy-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)pyridin-4-yl)thioureido)butyl)-54-(4-iodophenyl)-12,15,43,51-tetraoxo-3,6,9,19,22,25,28,31,34,37,40-undecaoxa-13,16,44,50-tetraazatetrapentacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (macropa-RPS-074)

Compound 408 (33 mg, 16 μmol) was dissolved in CH₂Cl₂ (2 mL). Then TFA(0.5 mL) was added and the reaction was stirred overnight at roomtemperature. The solvent was removed under N₂ flow and the crude productwas lyophilized to give a white residue (22 mg, 83%). Mass (ESI+): 832.0[(M+2H)/2]⁺. Calc. Mass: 1661.8. To a solution of the free amine (13 mg,7.8 μmol) and TEA (0.22 mL, 1.56 mmol) in DMF (1 mL) was added asolution of macropa-NCS (6 mg, 10 μmol) in DMF (1 mL). The resultingmixture was stirred for 90 min at room temperature. The reaction wasconcentrated under reduced pressure and the crude product was purifiedby prep HPLC. The peak corresponding to the desired product wascollected and lyophilized to give macropa-RPS-074 as a white powder (4.5mg, 26%). Mass (ESI+): 1126.6 [(M+2H)/2]⁺. Calc. Mass: 2251.3.

Synthesis of DOTA-Lys-IPBA

Preparation of 2,5-dioxopyrrolidin-yl 4-(4-iodophenyl)butanoate (409). Asolution of 4-(4-iodophenyl)butanoic acid (1.16 g, 4.0 mmol),N-hydroxysuccinimide (483 mg, 4.2 mmol), EDC.HCl (768 mg, 4.0 mmol) and4-DMAP (5.8 mg, 47 μmol) in CH₂₂ (30 mL) was stirred for 20 h. Then thereactionmixture was washed successively with 1M HCl, saturated NaHCesand brine. The organic layer was dried over MgS4, filtered andconcentrated under reduced pressure to give NHS ester 409 as a whitepowder (1.29 g, 83%). ¹H NMR (CDCl₃, 500 MHz): δ 7.61 (d, 2H, J=7.2 Hz).6.95 (d, 2H, J=7.6 Hz), 2.83 (s, 4H), 2.67 (t, 2H, J=7.6 Hz), 2.59 (t,2H, J=7.3 Hz), 2.03 (quint, 2H, J=7.3 Hz).

Preparation ofN²-(tert-butoxycarbonyl)-N⁶-(4-(4-iodophenyl)butanoyl)-L-lysine (410).Boc-L-Lys-OH (871 mg, 3.53 mmol) was suspended in DMF (10 mL) andstirred at room temperature. To the stirred suspension was slowly addeda solution of NHS ester 409 (1.29 g, 3.33 mmol) and NEt₃ (557 μL, 4.00mmol) in DMF (5 mL). The resulting suspension was stirred overnight atroom temperature. The reaction was quenched with 1M HCl (2 mL), and thesolvent was removed under reduced pressure. The crude residue wasdissolved in CH₂Cl₂ and washed successively with 1M HCl, saturatedNaHCO₃ solution and brine. The organic fraction was dried over MgSO₄,filtered and concentrated under reduced pressure to give Boc-Lys-IPBA(410) as a clear foam (1.25 g, 72%). ¹H NMR (CDCl₃, 500 MHz): δ 7.57 (d,2H, J=7.7 Hz), 6.91 (d, 2H, J=7.8 Hz), 5.94 (br s, 1H), 5.32 (br s, 1H),4.21 (m, 1H), 3.21 (m, 2H), 2.56 (t, 2H, J=7.6 Hz), 2.15 (t, 2H, J=7.1Hz), 1.90 (quint, 2H, J=7.5 Hz), 1.88 (m, 1H), 1.69 (m, 1H), 1.51 (m,2H), 1.42 (s, 9H), 1.41 (m, 2H). Mass (ESI+): 519.3 (M+H)+. Calc. Mass:518.4.

Preparation of N⁶-(4-(4-iodophenyl)butanoyl)-L-lysine (411).Boc-Lys-IPBA (518 mg, 1.0 mmol) was dissolved in 10 mL of a 20% v/vTFA/CH₂Cl₂ solution and stirred overnight at room temperature. Thesolvents were removed under a stream of N₂ and Lys-IPBA (411) wasisolated as a colorless oil (402 mg; 96%). ¹H NMR (DMSO, 500 MHz): δ7.75 (br s, 1H), 7.61 (d, 2H, J=7.8 Hz), 6.99 (d, 2H, J=7.8 Hz), 3.79(m, 1H), 2.99 (m, 2H), 2.02 (t, 2H, J=7.3 Hz), 1.74 (quint, 2H, J=7.4Hz), 1.37 (m, 4H), 1.24 (m, 2H). Mass (ESI+): 419.2 (M+H)+. Calc. Mass:418.3.

Preparation of2,2′,2″,2″′-(2-(4-(3-((S)-1-carboxy-5-(4-(4-iodophenyl)butanamido)pentyl)thioureido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraaceticacid (DOTA-Lys-IPBA). To a solution of Lys-IPBA (11 mg, 26 μmol) andDIPEA (17 μL, 100 μmol) in DMF (1 mL) was added a solution ofp-SCN-Bn-DOTA 2.5Cl_(2.5)H₂O (8 mg, 11.6 μmol) in H₂O (1 mL). Thereaction was stirred at room temperature for 4 h. The solvent wasremoved under reduced pressure and the crude residue was purified byprep HPLC. The peak corresponding to the product was collected andlyophilized to give DOTA-Lys-IPBA as a white powder (5 mg, 43%). ¹H NMR(DMSO, 500 MHz): δ 9.72 (br s, 1H), 7.89 (d, 2H, J=7.6 Hz), 7.77 (m,1H), 7.61 (d, 2H, J=7.1 Hz), 7.51 (d, 2H, J=7.8 Hz), 7.24 (m, 2H), 6.99(d, 2H, J=7.8 Hz), 4.84 (m, 1H), 3.70-3.04 (m, 14H), 3.01 (m, 4H), 2.02(t, 2H, J=7.1 Hz), 1.76 (m, 4H), 1.39 (m, 2H), 1.31 (m, 2H). Mass(ESI+): 971.0 (M+H)+. Calc. Mass: 969.9.

Radiochemistry. All reagents were purchased from Sigma Aldrich unlessotherwise noted, and were reagent grade. Hydrochloric acid (HCl) wastraceSELECT© (>99.999%) for trace analysis quality. Aluminum-backedsilica thin layer chromatography (TLC) plates were purchased from SigmaAldrich. Stock solutions of 0.05M HCl and 1M NH₄₀Ac were prepared bydilution in Milli-Q® water.

²²⁵Ac-RPS-074: To a solution of ²²⁵Ac(NO₃)₃ (Oak Ridge NationalLaboratory, USA) in 0.05M HCl (16.7-21.0 MBq in 950 μL) was added 20 μLof a 1 mg/mL solution of RPS-074 in DMSO. The pH was increased to 5-5.5by addition of 90 μL M NH₄₀Ac. The reaction was gently shaken for 20 minat 25° C. on an Eppendorf Thermomixer® C (VWR). Then the reaction wasdiluted with H₂O (9 mL) and passed through a pre-activated Sep-Pak C18Light cartridge (Waters). The reaction vial and cartridge were washedwith H₂O (5 mL) and the product was eluted with 500 μL of EtOH followedby 500 μL normal saline (0.9% NaCl in deionized H₂O; VWR). The eluatewas diluted to 4 mL in normal saline to give a stock solution with aradioactivity concentration of 1.1-1.5 MBq/mL. An aliquot was removedfrom the final solution and spotted onto an aluminum-backed silica TLCplate to determine radiochemical impurity. An aliquot of the ²²⁵Ac(NO₃)₃solution in 0.05M HCl was spotted in a parallel lane as a control. Theplate was immediately run in a 10% v/v MeOH/10 mM EDTA mobile phase, andthen allowed to stand for 8 h to enable radiochemical equilibrium to bereached. The plate was visualized on a Cyclone Plus Storage PhosphorSystem (Perkin Elmer) following a 3 min exposure on the phosphor screen.The radiochemical purity was expressed as a ratio of ²²⁵Ac-RPS-074 tototal activity and was determined to be 98.1%. The plate was visualizedagain 16 h later to confirm purity.

²²sAc-DOTA-Lys-IPBA: To a solution of ²²⁵Ac(NO₃)₃ (Oak Ridge NationalLaboratory, USA) in 0.05M HCl (5.0 MBq in 900 μL) was added 30 μL of a 1mg/mL solution of DOTA-Lys-IPBA in DMSO. The pH was increased to 5-5.5by addition of 80 μL 1M NH₄₀Ac, and the reaction was heated for 25 minat 95° C. on an Eppendorf Thermomixer® C (VWR). Then the reactionmixture was diluted with H2O (9 mL) and passed through a pre-activatedSep-Pak C18 Light cartridge (Waters). The reaction vial and cartridgewere washed with H2O (5 mL) and the product was eluted with 200 μL of a50% v/v EtOH/saline solution followed by 800 μL normal saline (0.9% NaClin deionized H2O; VWR). Radiochemical purity (96%) was determined byradioTLC as described above.

Cell Culture. The PSMA expressing human prostate cancer cell line,LNCaP, was obtained from the American Type Culture Collection. Cellculture supplies were obtained from Invitrogen unless otherwise noted.LNCaP cells were maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum (Hyclone), 4 mM L-glutamine, 1 mM sodium pyruvate, 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5 mg/mLD-glucose, and 50 μg/mL gentamicin in a humidified incubator at 37°C./5% CO₂. Cells were removed from flasks for passage or for transfer to12-well assay plates by incubating them with 0.25%trypsin/ethylenediaminetetraacetic acid (EDTA).

In vitro determination of IC₅₀. IC₅₀ values of the non-labeled,metal-free ligands were determined by screening in a multi-concentrationcompetitive binding assay against^(99m)Tc-((7S,12S,16S)-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-2-((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)-9,14-dioxo-2,8,13,15-tetraazaoctadecane-7,12,16,18-tetracarboxylicacid technetium tricarbonyl complex) (^(99m)Tc-MIP-1427), withK_(d)=0.64±0.46 nM [1] for binding to PSMA on LNCaP cells, according topreviously described methods [2] with small modifications. Briefly,LNCaP cells were plated 72 h prior to the experiment to achieve adensity of approximately 5×10⁵ cells/well (in triplicate) in RPMI-1640medium supplemented with 0.25% bovine serum albumin. The cells wereincubated for 2 h with 1 nM ^(99m)Tc-MIP-1427 in RPMI-1640 mediumcontaining 0.00125% w/v bovine serum albumin [3] in the presence of0.001-10,000 nM test compounds. Radioactive incubation media was thenremoved by pipette and the cells were washed twice using 1 mL ice-coldPBS 1× solution. Cells were harvested from the plates followingtreatment with 1 mL 1M NaOH and transferred to tubes for radioactivecounting using a 2470 Wizard² Automatic Gamma Counter (Perkin Elmer).Standard solutions (10% of activity added to each well) were prepared toenable decay correction. Cell-specific activity was corrected fornon-specific binding of ⁹⁹mTc-MIP-1427. IC₅₀ values were determined byfitting the data points to a sigmoidal Hills1 curve in Origin software.

Inoculation of mice with xenografts. All animal studies were approved bythe Institutional Animal Care and Use Committee of Weill CornellMedicine and were undertaken in accordance with the guidelines set forthby the USPHS Policy on Humane Care and Use of Laboratory Animals.Animals were housed under standard conditions in approved facilitieswith 12 h light/dark cycles. Food and water was provided ad libitumthroughout the course of the studies. Male BALB/c athymic nu/nu micewere purchased from the Jackson Laboratory. For inoculation in mice,LNCaP cells were suspended at 4×10⁷ cells/mL in a 1:1 mixture ofPBS:Matrigel (BD Biosciences). Each mouse was injected in the left flankwith 0.25 mL of the cell suspension. Biodistributions were conductedwhen tumors were in the range 200-800 mm³, while therapy studies wereinitiated when tumors were in the range 50-900 mm³.

Biodistribution studies in LNCaP xenograft mice. LNCaP xenografttumor-bearing mice (4 per time point per compound) were injectedintravenously with a bolus injection of 105 kBq and 320 ng (142 μmol) of²²⁵Ac-RPS-074. The mice were sacrificed at 4 h, 24 h, 7 d, 14 d and 21 dpost injection. A blood sample was removed, and a full biodistributionstudy was conducted on the following organs (with contents): heart,lungs, liver, small intestine, large intestine, stomach, spleen,pancreas, kidneys, muscle, bone and tumor. Tissues were weighed andcounted on a 2470 Wizard Automatic Gamma Counter (Perkin Elmer). Countswere corrected for decay and for activity injected, and tissue uptakewas expressed as percent injected dose per gram (% ID/g). Standard errormeasurement was calculated for each data point.

Therapy study in LNCaP xenograft mice. LNCaP xenograft tumor-bearingmice were randomly assigned to 5 groups (7 mice per group). One groupwas injected intravenously with a bolus injection of 148 kBq and 93 ng(41 μmol) ²²⁵Ac-RPS-074. The second treatment group was injected with 74kBq and 47 ng (21 μmol) ²²⁵Ac-RPS-074. The third treatment group wasinjected with 37 kBq and 23 ng (10 μmol) ²²⁵Ac-RPS-074. The fourth groupwas injected with the same volume of vehicle. The fifth group wasinjected with 133 kBq ²²⁵Ac-DOTA-Lys-IPBA. Tumor dimensions weremeasured and recorded three times weekly with digital calipers, andtumor volumes were calculated using the modified ellipsoid equation,V=0.5*length*width*width [4]. Mice were sacrificed after tumors reached2000 mm³ or if they showed any visible signs of distress, including lossof body weight, lack of appetite, excessive lethargy or formation ofsores and rashes. Body weight was measured with a digital balance twiceweekly, and mice were monitored for signs of distress. The mice werephotographed weekly to visually confirm changes in tumor volume.

Imaging of Treated Mice by μPET/CT. ⁶⁸Ga-PSMA-11 (also known as⁶⁸Ga-HBED-CC) was prepared as previously reported [5]. Eight mice wereinjected intravenously with 5.5 MBq ⁶⁸Ga-PSMA-11, 75 days afterinjection of either 138 kBq or 74 kBq ²²⁵Ac-RPS-074. The mice wereimaged using PET/CT (Inveon™; Siemens Medical Solutions, Inc.) at 1 hpost-injection following inhalation anesthetization with isoflurane.Total acquisition time was 30 min. A CT scan was obtained immediatelybefore the acquisition for both anatomical co-registration andattenuation correction. Images were reconstructed using the Inveon™software supplied by the vendor.

In Vitro and In Vivo Evaluation of RPS-074

The IC₅₀ value of RPS-074 was determined in vitro using amulti-concentration competitive binding assay against ^(99m)Tc-MIP-1427,which displays affinity for PSMA on LNCaP cells. It was demonstratedthat the IC₅₀ of RPS-074 was 12.0±3.4 nM, a value that is consistentwith the reported PSMA affinities of structurally analogoustrifunctional ligands [6]. The biodistribution of RPS-074 was examinedin vivo in LNCaP xenograft tumor-bearing mice. Mice were injectedintravenously with a bolus injection of 105 kBq and 320 ng (142 μmol) of²²⁵Ac-RPS-074. The mice were sacrificed at 4 h, 24 h, 7 d, 14 d and 21 dpost injection. FIG. 12 demonstrates that uptake of ²²⁵Ac-RPS-074 wasevident in the blood (12.3±0.5% ID/g), the lungs (5.0±0.2% ID/g), thekidneys (6.7±0.4% ID/g) and the tumor (5.8±0.3% ID/g) at 4 h postinjection (p.i.). By 24 h p.i., the activity in non-target tissue,including kidneys (3.0±0.3% ID/g), cleared in concert with bloodclearance, while activity in the tumor increased to 12.7±1.5% ID/g (FIG.12). By 7 d p.i., activity in the tumor remained high (9.5±1.5% ID/g),while the activity in the blood and every other tissue was less than 1%ID/g (FIG. 12). Persistent tumor uptake (11.9±1.5% ID/g) was evident at14 d p.i., with all other tissues becoming largely indistinguishablefrom background. By 21 d p.i., an anti-tumor effect was evident, withonly 1 mouse still bearing a tumor. Notwithstanding the absence oftumors, activity in the non-target tissue remained indistinguishablefrom background.

²²⁵Ac-RPS-074 showed excellent complex stability over 3 weeks even whentumors were absent. Biodistribution studies demonstrated that noaccumulation of signal was evident in the liver or bone, two organs thattypically take up free ²²⁵Ac³+[7]. 2²⁵Ac-RPS-074 also demonstrated afavorable pharmacokinetic profile; the tumor-to-kidney andtumor-to-blood ratios rapidly favor the tumor. By 24 h p.i., thetumor-to-kidney ratio reached 4.3±0.7 while at 7 d and 14 d p.i. it is15.0±2.9 and 62.2±9.5, respectively. The tumor-to-blood ratio at thesame time points is 3.3±0.5, 137.5±30.4 and 995.8±139.7. Significantdifferences in the pharmacokinetic profile demonstrates that the doseabsorbed by each tissue will be different.

Therapeutic Evaluation in LNCaP Xenograft Mice

LNCaP xenograft were randomly assigned to 5 groups and treated with abolus injection of 148 kBq and 93 ng (41 μmol)²²⁵Ac-RPS-074, 74 kBq and47 ng (21 μmol) ²²⁵Ac-RPS-074, 37 kBq and 23 ng (10 μmol)²²⁵Ac-RPS-074,133 kBq ²²⁵Ac-DOTA-Lys-IPBA, or vehicle control. A significant antitumoreffect was observed in the mice treated with 138 kBq and 74 kBq of²²⁵Ac-RPS-074. In the 138 kBq treatment group, 6/7 (86%) of tumors werenot detectable (<0.5 mm³) at 75 d post injection, while 1/7 (14%) oftumors were not detectable in the 74 kBq group. The distribution ofinitial tumor volumes was 100-624 mm³ and 64-455 mm³ for the two groups,respectively (FIG. 13). Tumor volume decreased in the 74 kBq group foras much as 42 d post injection before 6/7 (86%) of tumor volumes beganto increase again. The absence of tumors was confirmed by μPET/CTimaging with ⁶⁸Ga-PSMA-11 (FIG. 14) prior to the collection of samplesfor pathology. Those tumors that re-emerged in the 74 kBq treatmentgroup were shown by imaging to express PSMA. Physiologic uptake was alsoevident in the kidneys and salivary glands.

FIG. 13 demonstrates that both the 37 kBq treatment group and thepositive control group, which received 133 kBq ²²⁵Ac-DOTA-Lys-IPBA,showed an initial effect relative to the vehicle group, but tumorvolumes increased from a starting volume of 99-331 mm³ and 233-859 mm³,respectively, to a final volume of greater than 2000 mm³. A cleardose-response was evident in this study. Up to 42 days p.i., the 74 kBqand 138 kBq treatment groups behaved similarly, but while the tumorvolume of 5/7 (71%) of mice in the former group was measured to be lessthan 1 mm³, the tumors progressively returned. In contrast, the tumorsof the mice in the 37 kBq treatment group appeared to grow at a similarrate to the untreated tumors.

Every mouse in the 138 kBq treatment group survived the 75 d study (FIG.15). In contrast, in each of the other groups at least one mouse wassacrificed prior to the termination of the study due to excessive tumorgrowth. The survival curves for the 37 kBq group and the 133 kBq²²⁵Ac-DOTA-Lys-IPBA positive control group are similar, with 100% ofmice surviving the first 21 d. In contrast, only 1/7 (14%) of theuntreated mice survived to this time point. No toxic effects werevisible in any of the groups. The variation in body weight during the 75day study was 92-106% of the original measurement. The remaining micewere sacrificed at 75 d post injection and the tumor (if present),kidneys, liver, parotid glands and sublingual glands were excised andexamined for evidence of damage.

Further Exemplary Compounds of the Present Technology

The following compounds of the present technology were synthesized andcharacterized via similar protocols and methods as described above.

Section 1.4 References

-   [1] Hillier S M, Maresca K P, Lu G, Merkin R D, Marquis J C,    Zimmerman C N, Eckelman W C, Joyal J L, Babich J W. ^(99m)Tc-Labeled    Small-Molecule Inhibitors of Prostate-Specific Membrane Antigen for    Molecular Imaging of Prostate Cancer. J Nucl Med. 2013; 54:1369-76.-   [2] Kelly J M, Amor-Coarasa A, Nikolopoulou A, Wustemann T, Barelli    P, Kim D, Williams C. Jr, Zheng X, Bi C, Hu B, Warren J D, Hage D S,    DiMagno S G, Babich J W. Dual-Target Binding Ligands with Modulated    Pharmacokinetics for Endoradiotherapy of Prostate Cancer. J Nucl    Med. 2017; 58:1442-1449.-   [3] Benešová M, Umbricht C A, Schibli R, Mller C. Albumin-Binding    PSMA Ligands: Optimization of the Tissue Distribution Profile. Mol    Pharm. 2018; 15:934-946.-   [4] Jensen M M, Jorgensen J T, Binderup T, Kjor A. Tumor volume in    subcutaneous mouse xenografts measured by microCT is more accurate    and reproducible than determined by ¹⁸F-FDG-microPET or external    caliper. BMC Med Imaging 2008; 8:16.-   [5] Amor-Coarasa A, Kelly J M, Gruca M, Nikolopoulou A,    Vallabhajosula S, Babich J W.-   Continuation of comprehensive quality control of the itG ⁶⁸Ge/⁶⁸Ga    generator and production of ⁶⁸Ga-DOTATOC and ⁶⁸Ga-PSMA-HBED-C C for    clinical research studies. Nucl Med Biol. 2017; 53:37-39.-   [6] Kelly J, Amor-Coarasa A, Ponnala S, Nikolopoulou A, Williams C.,    Jr, Schlyer D, Zhao Y,-   Kim D, Babich J W. Trifunctional PSMA-Targeting Constructs for    Prostate Cancer with Unprecedented Localization to LNCaP Tumors. Eur    J Nucl Med Mol Imaging 2018; In press.-   Miederer M, Scheinberg D A, McDevitt M R. Realizing the potential of    the Actinium-225 radionuclide generator in targeted alpha-particle    therapy applications. Adv Drug Deliv Rev. 2008; 60:1371-1382.

While certain embodiments have been illustrated and described, a personwith ordinary skill in the art, after reading the foregoingspecification, can effect changes, substitutions of equivalents andother types of alterations to the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, prodrugs, metabolites,tautomers or racemic mixtures thereof as set forth herein. Each aspectand embodiment described above can also have included or incorporatedtherewith such variations or aspects as disclosed in regard to any orall of the other aspects and embodiments.

The present technology is also not to be limited in terms of theparticular aspects described herein, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods within thescope of the present technology, in addition to those enumerated herein,will be apparent to those skilled in the art from the foregoingdescriptions. Such modifications and variations are intended to fallwithin the scope of the appended claims. It is to be understood thatthis present technology is not limited to particular methods, reagents,compounds, compositions, labeled compounds or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. Thus, it is intended that thespecification be considered as exemplary only with the breadth, scopeand spirit of the present technology indicated only by the appendedclaims, definitions therein and any equivalents thereof.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso form part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments (for example, journals, articles and/or textbooks) referred toin this specification are herein incorporated by reference as if eachindividual publication, patent application, issued patent, or otherdocument was specifically and individually indicated to be incorporatedby reference in its entirety. Definitions that are contained in textincorporated by reference are excluded to the extent that theycontradict definitions in this disclosure.

Other embodiments are set forth in the following claims, along with thefull scope of equivalents to which such claims are entitled.

What is claimed is:
 1. A compound comprising: a tumor-binding domain, analbumin-binding domain, and a cytocidal or cytostatic therapeutic agent,wherein the tumor-binding domain comprises an active site that is distalto and sterically unimpeded by the albumin-binding domain and thetherapeutic agent, and the relative affinity of the tumor-binding domainand the albumin-binding domain differ in specific affinity by a factorof at least 100 to about 10,000.
 2. The compound of claim 1, wherein thetumor-binding domain binds to a tumor associated molecular targetselected from one or more of a tumor-specific cell surface protein,prostate specific membrane antigen (PSMA), somatostatin peptidereceptor-2 (SSTR2), alphavbeta3 (αvβ3), alphavbeta6, a gastrin-releasingpeptide receptor, a seprase, fibroblast activation protein alpha(FAP-alpha), an incretin receptor, a glucose-dependent insulinotropicpolypeptide receptor, VIP-1, NPY, a folate receptor, LHRH, a neuronaltransporter (e.g., noradrenaline transporter (NET)), EGFR, HER-2, VGFR,MUC-1, CEA, MUC-4, ED2,TF-antigen, an endothelial specific marker,neuropeptide Y, uPAR, TAG-72, a claudin, a CCK analog, VIP, bombesin,VEGFR, a tumor-specific cell surface protein, GLP-1, CXCR4, Hepsin,TMPRSS2, a caspace, cMET, or an overexpressed peptide receptor.
 3. Thecompound of claim 2, wherein the tumor-binding domain binds to the tumorassociated molecular target with moderate to high affinity.
 4. Thecompound of claim 1, wherein the cytocidal or cytostatic therapeuticagent is a toxin, a venom, a metabolic poison, a chemotherapeutic agent,an auger electron-emitting radionuclide, a beta-emitting radionuclide,or an alpha-emitting radionuclide.
 5. A compound comprising: amulti-targeted agent having a plurality of sterically unimpededtargeting domains, comprising a first targeting domain comprising ablood-protein binding domain having specific affinity for binding humanserum albumin in the range of about 0.25 to 50 micromolar, and a secondtargeting domain comprising a tumor-binding domain having specificaffinity for a tumor associated molecular target in the range of about0.1 to 75 nanomolar; wherein the relative affinities of the first andsecond targeting domains differ in specific affinity by a factor of atleast 100 to about 10,000; and a therapeutic domain comprising acytocidal or cytostatic therapeutic agent.
 6. The compound of claim 5,wherein the tumor associated molecular target is selected from one ormore of a tumor-specific cell surface protein, prostate specificmembrane antigen (PSMA), somatostatin peptide receptor-2 (SSTR2),alphavbeta3 (αvβ3), alphavbeta6, a gastrin-releasing peptide receptor, aseprase, fibroblast activation protein alpha (FAP-alpha), an incretinreceptor, a glucose-dependent insulinotropic polypeptide receptor,VIP-1, NPY, a folate receptor, LHRH, a neuronal transporter (e.g.,noradrenaline transporter (NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4,ED2,TF-antigen, an endothelial specific marker, neuropeptide Y, uPAR,TAG-72, a claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specificcell surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, oran overexpressed peptide receptor.
 7. The compound of claim 6, whereinthe tumor-binding domain binds to the tumor associated molecular targetwith moderate to high affinity.
 8. The compound of claim 5, wherein thecytocidal or cytostatic therapeutic agent is a toxin, a venom, ametabolic poison, a chemotherapeutic agent, an auger electron-emittingradionuclide, a beta-emitting radionuclide, or an alpha-emittingradionuclide.
 9. The compound of claim 5, wherein the therapeutic domaincomprises a covalently conjugated chelating agent or a covalentlyconjugated polyaza polycarboxylic macrocycle.
 10. The compound of claim9, wherein the therapeutic domain further comprises an augerelectron-emitting radionuclide, a beta-emitting radionuclide, or analpha-emitting radionuclide.
 11. A compound comprising: a multi-targetedagent having a plurality of sterically unimpeded targeting domainscomprising, a first targeting domain comprising a blood-protein bindingdomain having specific affinity for binding human serum albumin in therange of about 0.25 to 50 micromolar, a second targeting domaincomprising a tumor-binding domain having specific affinity forfibroblast activation protein alpha (FAP-alpha) in the range of about0.1 to 75 nanomolar; wherein the relative affinities of the first andsecond targeting domains differ in specific affinity by a factor of atleast 100 to about 10,000; and a therapeutic domain comprising acytocidal or cytostatic therapeutic agent.
 12. The compound of claim 11,wherein the blood-protein binding domain binds human serum albumin withan affinity in the range of about 0.4 to 20 micromolar, and thetumor-binding domain binds FAP-alpha with an affinity in the range ofabout 0.1 to 15 nanomolar; wherein the relative affinities of the firstand second targeting domains differ in specific affinity by a factor ofabout 1,000 to about 10,000.
 13. The compound of claim 11, wherein theblood-protein binding domain is selected from one or more of myristicacid, a substituted or unsubstituted indole-2-carboxylic acid, asubstituted or unsubstituted thioamide, a substituted or unsubstituted4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid, a substitutedor unsubstituted naphthalene acylsulfonamide, a substituted orunsubstituted diphenylcyclohexanol phosphate ester, a substituted orunsubstituted 4-iodophenylalkanoic acid, a substituted or unsubstituted3-(4-iodophenyl)propionic acid, a substituted or unsubstituted2-(4-iodophenyl)acetic acid, or a substituted or unsubstituted4-(4-iodophenyl)butanoic acid.
 14. The compound of claim 11, wherein thetumor-binding domain binds to the FAP-alpha with moderate to highaffinity.
 15. The compound of claim 11, wherein the cytocidal orcytostatic therapeutic agent is a toxin, a venom, a metabolic poison, achemotherapeutic agent, an auger electron-emitting radionuclide, abeta-emitting radionuclide, or an alpha-emitting radionuclide.
 16. Thecompound of claim 11, wherein the therapeutic domain comprises acovalently conjugated chelating agent or a covalently conjugated polyazapolycarboxylic macrocycle.
 17. The compound of claim 16, wherein thetherapeutic domain further comprises an auger electron-emittingradionuclide, a beta-emitting radionuclide, or an alpha-emittingradionuclide.